53BP2 complexes

ABSTRACT

The present invention relates to complexes of the 53BP2 protein with proteins identified as interacting with 53BP2 by a yeast two hybrid assay system. The proteins identified to interact with 53BP2 are β-tubulin, p62, hnRNP G, and three gene products, 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 encoded, in part, by the EST R72810 sequence. Thus, the invention provides complexes of 53BP2 and β-tubulin, p62, hnRNP G, 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 and derivatives, fragments and analogs thereof. The invention also provides the 53BP2-IP1, 53BP2-IP2 and 53BP2-IP3 genes and proteins and derivatives, fragments and analogs thereof. Methods of screening the complexes for efficacy in treating and/or preventing certain diseases and disorders, particularly cancer, autoimmune disease and neurodegenerative disease are also provided.

RELATED APPLICATIONS

This application is a divisional application under 37 C.F.R. §1.53(b) ofU.S. Ser. No. 08/935,450, filed on Sep. 23, 1997, now U.S. Pat. No.5,977,311, incorporated herein by reference in its entirety.

This invention was made with United States Government support underaward number 70NANB5H1066 awarded by the National Institute of Standardsand Technology. The United States Government has certain rights in theinvention.

FIELD OF THE INVENTION

The field of this invention is the complexes of 53BP2 protein with otherproteins, in particular, complexes of 53BP2 with β-tubulin, 53BP2 withp62, 53BP2 with hnRNP G, 53BP2 with 53BP2-IP1, 53BP2 with 53BP2-IP2, and53BP2 with 53BP2-IP3 proteins. The invention includes antibodies to53BP2 complexes, and their use in, inter alia, screening, diagnosis,prognosis and therapy. The invention further relates to 53BP2-IP1,53BP2-IP2, and 53BP2-IP3 genes and proteins and derivatives, fragmentsand analogs, thereof.

BACKGROUND OF THE INVENTION 53BP2

The human Bcl2/p53 binding protein, known as 53BP2, or BBP (GenBankAccession Number U58334, Naumovski and Cleary, 1996, Mol. Cell. Biol.16: 3884-3892) impedes cell cycle progression from G2 to M phase. 53BP2competes with Bcl2 for binding to p53, and thus is critical formodulation of p53 function (Naumovski and Cleary, 1996, Mol. Cell. Biol.16: 3884-3892). In turn, p53 is a critical tumor suppressor protein thatcan activate or repress transcription, and thus mediate cell cycleprogression (Murray and Hunt, 1993, The Cell Cycle: An Introduction, W.H. Freeman and Co., New York). 53BP2 binds to the central DNA bindingdomain of p53 via two adjacent ankryin repeats and an SH3 domain, thussupporting its ability to modulate, inter alia, p53 DNA binding andstability, and thus, its tumor suppression (Iwabuchi et al., 1994, Proc.Natl. Acad. Sci. USA 91: 6098-6102). Importantly, the most frequent p53mutations observed in human cancers map to the region of 53BP2 binding(Gorina and Pavletich, 1996, Science 274: 1001-1005). 53BP2 alsomodulates the dephosphorylation status, and thus the function of p53,via its binding to protein phosphatase 1 (PP1), thus inhibiting thelatter protein's ability to dephosphorylate p53 (Helps et al., 1995,FEBS Letts. 377: 295-300). Phosphorylation at multiple p53 sites affectsits transcriptional activation/inhibition, but in a complicated fashionthat is not easy to predict (Fuchs et al., 1995, Eur J Biochem 228:625-639; Hecker et al., 1996, Oncogene 12: 953-961). The above dataindicate that proteins that interact with 53BP2 have a fairly directmeans to modulate p53 function. It has been previously shown that onesuch protein, PP1, binds to the C-terminal region of 53BP2 containingthe critical ankyrin and SH3 binding domains (Helps et al., 1995, FEBSLetts. 377: 295-300). In sum, 53BP2 has likely roles in the control ofcell cycle progression, transcriptional regulation, cellular apoptosisand differentiation, intracellular signal transduction, andtumorigenesis.

β-Tubulin

Human β-tubulin (GenBank Accession No. X79535, Leffers et al., 1994,GenBank direct submission Jun. 1, 1994) exists in at least six differentisoforms that are expressed from separate genes in a tissue specificdistribution (Ranganathan et al., 1997, Prostate 30: 263-268). Tubulinsare critical to the enzymic-mechanical conversion of ATP hydrolysis tomolecular movement along microtubules. The C-terminus of β-tubulin, inparticular the last 12 amino acid residues, interacts with kinesinmotors to modulate microtubule polymerization, dynamics, and drugsensitivity (Ranganathan et al., 1997, Prostate 30: 263-268; Tucker andGoldstein, 1997, J. Biol. Chem. 272: 9481-9488). This function may havepathophysiological significance; type IV β-tubulin is highly expressedin adenocarcinomas of the prostate, and type II β-tubulin expression isup-regulated in adenocarcinomas that become malignant (Ranganathan etal., 1997, Prostate 30: 263-268). Colchicine, which specificallyinteracts with β-tubulins to arrest cellular outgrowth, is an effectiveantitumor agent (Banerjee, 1997, Biochem. Biophys. Res. Commun. 231:698-700). Further, microtubules, possibly through beta-tubulin bindingto proteins that contain Src homology 2 (SH2) domains, play importantroles in the assembly of signaling molecular complexes involved incellular transformation (Itoh et al., 1996, J. Biol. Chem. 217:27931-27935). In summary, β-tubulin has roles in tumorigenesis and tumorprogression, cell structure and intracellular protein transport, celldifferentiation, and intracellular signalling.

p62

Human p62 (GenBank Accession No. M88108, Wong et al., 1992, Cell 69:551-558) is a 62 kD tyrosine phosphoprotein that displays significanthomology to the hnRNP protein GRP33. p62 associates with thep21^(waf)GTPase-activating protein (GAP). The binding depends on thephosphorylation state of p62 and occurs via SH2 domains in GAP (Wong etal., 1992, Cell 69: 551-558). The protein p62 further associates withSrc family tyrosine kinase SH3 domains in signalling proteins. Since p62can interact with multiple proteins at once via its several SH3 bindingdomains, p62 serves to physically link Src kinase activity withdownstream effectors such as GRB2 and phospholipase C gamma-1 (Richardet al., 1995, Mol. Cell. Biol. 15: 186-197). In its dephosphorylatedstate, p62 actively binds RNA via a “KH domain” (Wang et al., 1995, J.Biol. Chem. 270: 2010-2013). Phosphorylation severely impairs p62binding to RNA, suggesting that p62 RNA binding is regulated in vivo.p62 is known to specifically interact with ubiquitin via its C-terminal80 residues (Vadlamudi et al., 1996, J. Biol. Chem. 271: 20235-20237),thus implicating p62 in ubiquitin-mediated proteolysis. It alsospecifically interacts with CSK, a cytosolic protein tyrosine kinasethat negatively regulates Src family protein tyrosine kinases, and it ishypothesized that this binding mediates docking of proteins, includingGAP and CSK, to cytoskeletal and membrane regions upon c-Src activation(Neet and Hunter, 1995, Mol. Cell. Biol. 15: 4908-4920). Levels ofphospho-p62, detected by Western blotting, increase markedly in v-abltransformed lymphoblasts (a cell model of leukemia) that reach advancedstages of feeder-layer independent agar growth (Clark and Liang, 1995,Leukemia 9: 165-174). In summary, p62 is implicated in celltransformation and tumor progression, intracellular signalling andcellular activation by c-Src, ubiquitin-mediated proteolysis, and mRNAbinding and metabolism.

hnRNP G

Human hnRNP G protein (GenBank Accession No. Z23064; Soulard et al.,1993, Nucleic Acids Res. 21: 4210-4217) is an RNA binding protein whosehomolog (p43) was originally identified as an autoantigenic nuclearprotein in dogs with a lupus-like syndrome. It is a glycosylatedcomponent of heterogenous nuclear ribonucleoprotein complexes thatcontains an RNA binding domain at its amino terminus and a carboxyldomain rich in serines, arginines, and glycines (Soulard et al., 1993,Nucleic Acids Res. 21: 4210-4217). Likely roles for hnRNP G includeregulation of cell division, translational, and transcription. It mayalso function in various autoimmune diseases, such as systemic lupuserythematosus and rheumatoid arthritis.

The newly identified 53BP2-IP1, 53BP2-IP2 and 53BP2-IP3 proteins areencoded in part by a nucleotide sequence identified as EST R72810 in theGenBank Database (Hillier et al., 1995, GenBank Direct Submission Jun.2, 1995, Accession No. 157775) obtained from the Soares (human) breastlibrary 2NbHBst. Over a span of 54 nucleotides, the EST R72810 sequencedisplays 74% identity to the Simian immunodeficiency virus SIVpt5 gene(GenBank Accession No. U05129), but otherwise displays no significanthomology to known proteins.

53BP2 complexes with any of β-tubulin, p62, hnRNP G, 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 have not been previously described.

Citation of a reference herein shall not be construed as an admissionthat such is prior art to the present invention.

SUMMARY OF THE INVENTION

The present invention provides certain compositions and methods ofproduction of protein complexes of 53BP2 with proteins that interactwith (i.e., bind to) 53BP2 (the proteins shown to bind with 53BP2 aredesignated “53BP2-IP” for 53BP2 interacting protein, and the complexesof 53BP2 and a 53BP2-IP are designated as 53BP2:53BP2-IP herein).Specifically, the invention relates to complexes of 53BP2, andderivatives, fragments and analogs of 53BP2 with β-tubulin, with p62,with hnRNP G, with 53BP2-IP1, with 53BP2-IP2 and with 53BP2-IP3, andtheir derivatives, analogs and fragments. The present invention furtherprovides methods of screening for proteins that interact with 53BP2, orderivatives, fragments or analogs, thereof; preferably the method ofscreening is a yeast two hybrid assay system or a variation thereof.

The invention further relates to nucleotide sequences of 53BP2-IP1,53BP2-IP2 and 53BP2-IP3 genes (human 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3genes and homologs of other species), as well as derivatives (e.g.,fragments) and analogs thereof. Nucleic acids hybridizable to orcomplementary to the foregoing nucleotide sequence, such as the inversecomplement (i.e. has the complementary sequence running in reverseorientation to the strand so that the inverse complement would hybridizewithout mismatches to the nucleic acid strand; thus, for example, wherethe coding strand is hybridizable to a nucleic acid with no mismatchesbetween the coding strand and the hybridizable strand, then the inversecomplement of the hybridizable strand is identical to the coding strand)of the foregoing sequences, are provided. The invention also relates to53BP2-IP1, 53BP2-IP2 and 53BP2-IP3 derivatives and analogs of theinvention that are functionally active, i.e., they are capable ofdisplaying one or more known functional activities of a wild-type53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 protein. Such functional activitiesinclude, but are not limited to ability to bind with [or compete forbinding with] 53BP2, antigenicity [ability to bind (or compete with53BP2-IP1, 53BP2-IP2 or 53BP2-IP3 for binding) to an anti-53BP2-IP1,anti-53BP2-IP2 or anti-53BP2-IP3 antibody, respectively], andimmunogenicity (ability to generate antibody which binds 53BP2-IP1,53BP2-IP2, or 53BP2-IP3, respectively).

Methods of production of the 53BP2:53BP2-IP complexes and of 53BP2-IP1,53BP2-IP2, and 53BP2-IP3 proteins, and derivatives and analogs of thecomplexes and proteins, e.g., by recombinant means, are also provided.Pharmaceutical compositions are also provided.

The invention further provides methods of modulating (i.e., inhibitingor enhancing) the activity of 53BP2:53BP2-IP complexes, particularly53BP2:β-tubulin, 53BP2:p62, 53BP2:hnRNP G, 53BP2:53BP2-IP1,53BP2:53BP2-IP2, or 53BP2:53BP2-IP3 complexes. The protein components ofthe complexes have been implicated in cellular functions, including butnot limited to: control of cell cycle progression, cellulardifferentiation and apoptosis, tumorigenesis and tumor progression;regulation of transcription and translation; control of intracellularsignal transduction, including c-Src signalling; control ofubiquitin-mediated protein degradation, and processing involving mRNAbinding and stability. Accordingly, the invention provides methods ofscreening 53BP2:53BP2-IP complexes, particularly complexes of 53BP2 withβ-tubulin, p62, hnRNP G, 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 and the53BP2-IP1, 53BP2-IP2 and 53BP2-IP3 proteins, as well as derivatives andanalogs of the 53BP2:53BP2-IP complexes and 53BP2-IP1, 53BP2-IP2 and53BP2-IP3 proteins for the ability to alter cell functions, particularlythose cell functions in which 53BP2 and/or a 53BP2-IP has beenimplicated, such as but not limited to, cell proliferation,differentiation and apoptosis, tumorigenesis and cell transformation,intracellular signal transduction, gene expression, ubiquitin-mediatedprotein degradation, and mRNA stability.

The present invention also relates to therapeutic and prophylactic aswell as diagnostic, prognostic, and screening methods and compositionsbased upon 53BP2:53BP2-IP complexes (and the nucleic acids encoding theindividual proteins that participate in the complexes) as well as53BP2-IP1 and 53BP2-IP2 and 53BP2-IP3 proteins and nucleic acids.Therapeutic compounds of the invention include, but are not limited to,53BP2:53BP2-IP complexes and complexes where one or both members of thecomplex is a derivative or analog of 53BP2 or 53BP2-IP; 53BP2-IP1,53BP2-IP2 and 53BP2-IP3 proteins and derivatives, fragments and analogsthereof; antibodies to and nucleic acids encoding the foregoing; andantisense nucleic acids to the nucleotide sequences encoding the complexcomponents and 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 antisense nucleicacids. Diagnostic, prognostic and screening kits are also provided.

Animal models and methods of screening for modulators (i.e. agonists,antagonists and inhibitors) of the activity of 53BP2:53BP2-IP complexesand 53BP2-IP1, 53BP2-IP2 and 53BP2-IP3 proteins are also provided.

Methods of identifying molecules that inhibit, or alternatively, thatincrease formation of 53BP2:53BP2-IP complexes are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The nucleotide sequence of 53BP2 (GenBank Accession No. U58334(SEQ ID NO: 1)) and deduced amino acid sequence (SEQ ID NO:2). Theamino-terminal start site of the sequence used as bait in the assaysdescribed in Section 6, infra, is indicated by the arrow labeled “A”.

FIG. 2. The nucleotide sequence (SEQ ID NO:3) and amino acid sequence(SEQ ID NO:4) of β-tubulin (GenBank Accession No. X79535). Prey sequenceA begins at base 820 (and amino acid 253) at the arrow labeled “A”. Preysequence B begins at base 895 (and amino acid 278) at the arrow labeled“B”.

FIG. 3. The nucleotide sequence and corresponding amino acid sequence ofthe p62 protein (GenBank Accession No. M88108 (SEQ ID NOS:5 and 6,respectively)). The amino-terminal start site of the prey sequenceidentified in the assay described in Section 6, infra, is indicated bythe arrow labeled “A”.

FIG. 4. The hnRNP G nucleotide acid sequence (SEQ ID NO:7) and aminoacid sequence (SEQ ID NO:8). The amino-terminal start site of the preysequence identified in the experiments described in Section 6, infra, isindicated by the arrow labeled “A”.

FIG. 5. The nucleotide sequence of EST R72810 (SEQ ID NO:9). The entiresequence is the prey sequence identified in Section 6 infra.

FIG. 6. Schematic of the portions of 53BP2, β-tubulin, p62, hnRNP G, and53BP2-IP1/IP2/IP3 (i.e., the proteins potentially encoded, at least inpart, by the extended EST R72810 sequence) that interact in a 53BP2:IPcomplex in the yeast two hybrid assay system. The sequences of 53BP2,β-tubulin, p62, hnRNP G and EST R78210 proteins are depicted as bars,with the starting and ending amino acid numbers (as depicted for eachprotein in FIGS. 1-4, 9 and 10A-F (SEQ ID NOS: 2, 4, 6, 8, 11, 12 and13, respectively). The portions of each sequence either used as bait (inthe case of 53BP2) or identified in the assay (“prey sequence”) (in thecase of β-tubulin, p62, hnRNP G and EST R78210 encoded proteins areblackened and the first amino acid number of the bait or prey sequence,as the case may be, is indicated above each bar. For β-tubulin, theN-terminal portion of a second, longer interacting sequence is indicatedby a horizontal line (with the first amino acid of this extensionindicated above the bar). For 53BP2-IP1 and 53BP2-IP3, the first aminoacid is denoted by “>1”, since the actual amino terminus is predicted toextend beyond the 5′ end of the assembled nucleotide sequence.

FIG. 7. Matrix of results of yeast two hybrid system assays. The resultsof assays using yeast expressing hybrids of the bait proteins B1, MDM2and 53BP2 are indicated in the rows designated B1, MDM2 and 53BP2 matedwith yeast cells expressing hybrids of the prey proteins P1, P2, PP1α,p62 and β-tubulin (“β-tub.”), as indicated by the rows designated as P1,P2, PP1α, p62 and β-tub, are depicted. A positive interaction for a baitand prey proteins is indicated as “+” in the box forming theintersection between the particular bait and prey proteins; a lack ofinteraction is designated by an empty box. Boxes labeled A, B, C and Dindicate the results of matings of yeast expressing B1 and PP1α, 53BP2and PP1α, 53BP2 and p62, and 53BP2 and β-tubulin, respectively.

FIG. 8. This figure illustrates the general procedure used to assemblethe longest possible contiguous nucleic acid sequence from a particularEST sequence. The starting EST nucleic acid sequence, shown as the linelabeled above as B, is run through the N.C.B.I. “BLAST” Program, andcompared to all sequences in the “nr” database. Sequences that alignwith 95% or greater identity at the nucleic acid level over theirtermini of at least 30 bases are utilized if the alignment will resultin 5′ extension (Sequence A) or 3′ extension (Sequence C) of thestarting EST sequence.

FIG. 9. The nucleotide sequence of EST R72810 and contiguous ESTsequences (SEQ ID NO:10) is depicted. The original EST R72810 sequenceis shown in bold lettering; a 5′ extension was achieved with EST C17385marked with underline; and 3′ extensions were made, first from ESTAA464793 indicated by boxed lettering, and second with EST AA479761indicated by bold, italic lettering. The 5′ of the prey interactingsequence denoted as “A”; the 3′ end of the sequence is indicated by astarred arrow.

FIGS. 10A-F. The predicted open reading frames and translation of theopen reading frames, in all three frames, of the nucleotide sequence ofSEQ ID NO:10. (A and B). The C-terminus of 53BP2-IP3 (SEQ ID NO:13) isencoded in Translation Frame +1 at the 5′ end of the assemblednucleotide sequence as depicted graphically in panel A. Panel B presentsthe nucleotide and predicted amino acid sequence of the C-terminus of53BP2-IP3. (C and D). 53BP2-IP2 (SEQ ID NO:12) is encoded in translationFrame +2 from nucleotides 44-652, as depicted in panel C. Panel Dpresents the nucleotide and predicted amino acid sequence of 53BP2-IP2.(E and F). 53BP2-IP1 (SEQ ID NO:11) is encoded in translation Frame +2from nucleotides 44-652, as depicted in panel E. Panel F presents thenucleotide and predicted amino acid sequence of 53BP2-IP1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the identification of proteins thatinteract with 53BP2 (termed herein “53BP2-IPs”) using an improved,modified form of the yeast two hybrid system. β-tubulin, p62, hnRNP G,and 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 were found to form complexesunder physiological conditions with 53BP2 (the complexes of 53BP2 with a53BP2-IP are indicated as “53BP2:53BP2-IP” complexes herein). These53BP2:53BP2-IP complexes, by virtue of the interaction, are implicatedin modulating the functional activities of 53BP2 and its bindingpartners. Such functional activities include, but are not limited to,cell cycle control, transcriptional regulation, cellular apoptosis anddifferentiation, intracellular signal transduction, tumorigenesis andtumor progression, protein transport and cell structure, celldifferentiation, cellular activation by c-Src, ubiquitin-mediatedproteolysis, mRNA binding and metabolism, translational regulation, andautoimmune disease.

The present invention relates to methods of screening for proteins thatinteract with (e.g. bind to) 53BP2. The invention further relates to53BP2 complexes, in particular 53BP2 complexes with one of the followingproteins: β-tubulin, p62, hnRNP G, 53BP2-IP1, 53BP2-IP2 or 53BP2-IP3.The invention further relates to complexes of 53BP2 or derivatives,analogs and fragments of 53BP2 with β-tubulin, p62, hnRNP G, 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 derivatives, analogs and fragments thereof. In apreferred embodiment such complexes bind an anti-53BP2:53BP2-IP complexantibody. In a specific embodiment, complexes of 53BP2 with a proteinthat is not protein phosphatase I alpha (PPI alpha) or p53 are provided.

The invention also provides methods of producing and/or isolating the53BP2:53BP2-IP complexes. In a specific embodiment, the inventionprovides methods of recombinantly expressing both 53BP2 and its bindingpartner (or fragments, derivatives or homologs of one or both members ofthe complex) either where both binding partners are under the control ofone heterologous promoter (i.e. a promoter not naturally associated withthe native gene encoding the particular complex component) or where eachis under the control of a separate heterologous promoter.

In another aspect, the invention provides the nucleotide sequences of53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 and their encoded proteins. Theinvention further relates to 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3proteins, derivatives, fragments and homologs thereof, as well asnucleic acids encoding the 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 proteins,derivatives, fragments and homologs. The invention provides 53BP2-IP1,53BP2-IP2, and 53BP2-IP3 proteins and genes encoding these proteins ofmany different species, particularly vertebrates, and more particularlymammals. In a preferred embodiment, the 53BP2-IP1, 53BP2-IP2 and53BP2-IP3 proteins and genes are of human origin. Production of theforegoing proteins and derivatives, e.g., by recombinant methods, isprovided.

The invention further relates to 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3derivatives and analogs which are functionally active, i.e., capable ofdisplaying one or more known functional activities associated with afull length (wild-type) 53BP2-1P1, 53BP2-IP2, and 53BP2-IP3. Suchfunctional activities include, but are not limited to, ability to form acomplex with 53BP2, antigenicity [ability to bind (or compete with53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 for binding) to an anti-53BP2-IP1,anti-53BP2-IP2, or anti-53BP2-IP3 antibody, respectively],immunogenicity (ability to generate an antibody that binds to 53BP2-IP1,53BP2-IP2, or 53BP2-IP3, respectively), etc.

Methods of diagnosis, prognosis, and screening for disease and disordersassociated with aberrant levels of 53BP2:53BP2-IP complexes or of53BP2-IP1, 53BP2-IP2 or 53BP2-IP3 are provided. The invention alsoprovides methods of treating or preventing diseases or disordersassociated with aberrant levels of 53BP2:53BP2-IP complexes or53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 or aberrant levels of the activity ofone or more of the components of the complex by administration of the53BP2:53BP2-IP complexes, 53BP2-IP1, 53BP2-IP2, 53BP2-IP3 or modulatorsof 53BP2:53BP2-IP complex formation or activity (e.g., antibodies thatbind the 53BP2:53BP2-IP complex, uncomplexed 53BP2 or its bindingpartner or a fragment thereof—preferably the fragment containing theportion of 53BP2 or the 53BP2-IP that is directly involved in complexformation), mutants of 53BP2 or the 53BP2-IP that increase or decreasebinding affinity, small molecule inhibitors/enhancers of complexformation, antibodies that either stabilize or neutralize the complex,etc.

Methods of assaying 53BP2:53BP2-IP complexes, 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 for activity as therapeutics or diagnostics as well as methodsof screening for 53BP2:53BP2-IP complex, 53BP2-IP1, 53BP2-IP2 or53BP2-IP3 modulators (i.e., inhibitors, agonists and antagonists) arealso provided.

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections whichfollow.

BP2:1P Complexes and 53BP2-IP1, 53BP2-IP2 and 53BP2-IP3 Proteins andDerivatives and Anologs

The invention provides 53BP2:53BP2-IP complexes, and, in particularaspects, complexes of 53BP2 and β-tubulin, 53BP2 and p62, 53BP2 andhnRNP G, 53BP2 and 53BP2-IP1, 53BP2 and 53BP2-IP2, and 53BP2 and53BP2-IP3. In a preferred embodiment, 53BP2 is complexed with a proteinthat is not a PP1 alpha protein or a p53 protein. In another preferredembodiment, the 53BP2:53BP2-IP complexes are complexes of humanproteins. The invention also relates to complexes of derivatives(including fragments) and analogs of 53BP2 with a 53BP2-IP, complexes of53BP2 with derivatives (including fragments) and analogs of a 53BP2-IP,and complexes of derivatives (including fragments) and analogs of 53BP2and a 53BP2-IP (as used herein, fragment, derivative or analog of a53BP2:53BP2-IP complex includes complexes where one or both members ofthe complex are fragments, derivatives or analogs of the wild-type 53BP2or 53BP2-IP protein). Preferably, the 53BP2:53BP2-IP complexes in whichone or both members of the complex are a fragment, derivative or analogof the wild type protein are functionally active 53BP2:53BP2-IPcomplexes. In particular aspects, the native proteins, derivatives oranalogs of 53BP2 and/or the 53BP2-IP are of animals, e.g. mouse, rat,pig, cow, dog, monkey, human, fly, frog, or of plants. “Functionallyactive 53BP2:53BP2-IP complex” as used herein refers to that materialdisplaying one or more known functional attributes of a complex of fulllength 53BP2 with a full length 53BP2-IP (e.g., β-tubulin, p62, hnRNP G,53BP2-IP1, 53BP2-IP2, or 53BP2-IP3) including but not exclusive to cellcycle control, modulation of cell apoptosis and differentiation, controlof transcriptional and translational regulation, effects onintracellular signal transduction, protein transport, and c-Srcactivation, effects on tumorigenesis and tumor progression,ubiquitin-mediated proteolysis, effects on mRNA binding and metabolism,binding to an anti-53BP2:53BP2-IP complex antibody, etc., and otheractivities as they are described in the art. For example, suchderivatives or analogs which have the desired immunogenicity orantigenicity can be used in immunoassays, for immunization, forinhibition of 53BP2:53BP2-IP complex activity, etc. Derivatives oranalogs that retain, or alternatively lack or inhibit, a property ofinterest (e.g., participation in a 53BP2:53BP2-IP complex) can be usedas inducers, or inhibitors, respectively, of such a property and itsphysiological correlates. A specific embodiment relates to a53BP2:53BP2-IP complex of a fragment of 53BP2 and/or a fragment of53BP2-IP that can be bound by an anti-53BP2 and/or 53BP2-IP antibody orantibody specific for a 53BP2:53BP2-IP complex when such a fragment isincluded within a 53BP2:53BP2-IP complex.

Fragments and other derivatives or analogs of 53BP2:53BP2-IP complexescan be tested for the desired activity by procedures known in the art,including but not limited to the assays described in Section 5.6.

In specific embodiments, the invention provides 53BP2:53BP2-IP complexescomprising fragments of one or both members of the complex. In apreferred embodiment, these fragments consist of, but are not exclusiveto, the C-terminal domain of 53BP2 of amino acid 704-1005 (as depictedin FIG. 1 (SEQ ID NO:2)), and fragments of β-tubulin, p62, hnRNP G,53BP2-IP1, 53BP2-IP2, or 53BP2-P3, of those regions identified asinteracting with 53BP2 in the yeast two hybrid assay (e.g., amino acids253-445 or 278-445 of β-tubulin as depicted in FIG. 2 (SEQ ID NO:4),amino acids 275-443 of p62 as depicted in FIG. 3 (SEQ ID NO:6), aminoacids 88-439 of hnRNP G as depicted in FIG. 4 (SEQ ID NO:8), amino acids8-173 of 53BP2-IP1 as depicted in FIG. 10F (SEQ ID NO:11), amino acids1-70 of 53BP2-IP2 as depicted in FIG. 10D (SEQ ID NO:12), and aminoacids 8-33 of 53BP2-IP3 as depicted in FIG. 10B (SEQ ID NO:13)).Fragments, or proteins comprising fragments, lacking some or all of theforegoing regions of either member of the complex assay, are alsoprovided. Nucleic acids encoding the foregoing are provided.

The invention further relates to 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3proteins as well as derivatives (including but not limited to fragments)and homologs and paralogs of 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3proteins. In one embodiment human 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3genes and proteins are provided. In specific aspects, the nativeproteins, fragments, derivatives or analogs of 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 are of animals, e.g. mouse, rat, pig, cow, dog, monkey, human,fly, frog, or of plants. In other specific embodiments, the fragment,derivative or analog is functionally active, i.e., capable of exhibitingone or more functional activities associated with full-length, wild-type53BP2-IP1, 53BP2-IP2, or 53BP2-IP3, e.g., ability to bind 53BP2,immunogenicity or antigenicity.

The nucleotide sequences encoding, and the corresponding amino acidsequences, of human 53BP2, β-tubulin, p62, and hnRNP G are known(GenBank Accession No. U58334; GenBank Accession No. X79535; GenBankAccession No. M88108; and GenBank Accession No. Z23064, respectively),and are provided in FIGS. 1-4, respectively (SEQ ID NOS:1, 3, 5, and 7,respectively). Nucleic acids encoding 53BP2, β-tubulin, p62 or hnRNP Gcan be obtained by any method known in the art, e.g, by PCRamplification using synthetic primers hybridizable to the 3′ and 5′ endsof the sequence and/or by cloning from a cDNA or genomic library usingan oligonucleotide specific for the gene sequence (e.g., as described inSection 5.2, infra). Homologs (e.g., nucleic acids encoding 53BP2,β-tubulin, p62, and hnRNP G of species other than human) or otherrelated sequences (e.g., paralogs) can be obtained by low, moderate orhigh stringency hybridization with all or a portion of the particularhuman sequence as a probe using methods well known in the art fornucleic acid hybridization and cloning (e.g., as described in Section5.2, infra, for 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 sequences).

The 53BP2, β-tubulin, p62, hnRNP G, and 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 proteins, either alone or in a complex, can be obtained bymethods well known in the art for protein purification and recombinantprotein expression. For recombinant expression of one or more of theproteins, the nucleic acid containing all or a portion of the nucleotidesequence encoding the protein can be inserted into an appropriateexpression vector, i.e., a vector that contains the necessary elementsfor the transcription and translation of the inserted protein codingsequence. The necessary transcriptional and translational signals canalso be supplied by the native promoter for 53BP2 or any 53BP2-IP genes,and/or their flanking regions.

A variety of host-vector systems may be utilized to express the proteincoding sequence. These include but are not limited to mammalian cellsystems infected with virus (e.g. vaccinia virus, adenovirus, etc.);insect cell systems infected with virus (e.g. baculovirus);microorganisms such as yeast containing yeast vectors; or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.

In a preferred embodiment, the 53BP2:53BP2-IP complexes are obtained byexpressing the entire 53BP2 sequence and a 53BP2-IP coding sequence inthe same cell, either under the control of the same promoter or twoseparate promoters. In yet another embodiment, a derivative, fragment orhomolog of 53BP2 and/or a derivative, fragment or homolog of a 53BP2-IPare recombinantly expressed. Preferably the derivative, fragment orhomolog of 53BP2 and/or the 53BP2-IP protein forms a complex with abinding partner identified by a binding assay, such as the modifiedyeast two hybrid system described in Section 5.7.1 infra, morepreferably forms a complex that binds to an anti-53BP2:53BP2-IP complexantibody.

Any of the methods described in Section 5.2 infra, for the insertion ofDNA fragments into a vector may be used to construct expression vectorscontaining a chimeric gene consisting of appropriatetranscriptional/translational control signals and protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of nucleic acid sequences encoding 53BP2 and a 53BP2-IP(e.g., β-tubulin, p62, hnRNP G, 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3), orderivatives, fragments or homologs thereof, may be regulated by a secondnucleic acid sequence so that the genes or fragments thereof areexpressed in a host transformed with the recombinant DNA molecule(s).For example, expression of the proteins may be controlled by anypromoter/enhancer known in the art. In a specific embodiment, thepromoter is not native to the genes for 53BP2 or the 53BP2-IP. Promoterswhich may be used include but are not limited to the SV40 early promoter(Bernoist and Chambon, 1981, Nature 290: 304-310), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamotoet al., 1980, Cell 22: 787-797), the herpes thymidine kinase promoter(Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1441-1445), theregulatory sequences of the metallothionein gene (Brinster et al., 1982,Nature 296: 39-42); prokaryotic expression vectors such as theβ-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad.Sci. USA 75: 3727-3731) or the tac promoter (DeBoer et al., 1983, Proc.Natl. Acad. Sci. USA 80: 21-25; see also “Useful Proteins fromRecombinant Bacteria”: in Scientific American 1980, 242:79-94); plantexpression vectors comprising the nopaline synthetase promoter(Herrar-Estrella et al., 1984, Nature 303: 209-213) or the cauliflowermosaic virus 35S RNA promoter (Garder et al., 1981, Nucleic Acids Res.9:2871), and the promoter of the photosynthetic enzyme ribulosebisphosphate carboxylase (Herrera-Estrella et al., 1984, Nature 310:115-120); promoter elements from yeast and other fungi such as the Gal4promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinasepromoter, the alkaline phosphatase promoter, and the following animaltranscriptional control regions that exhibit tissue specificity and havebeen utilized in transgenic animals: elastase I gene control regionwhich is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald 1987, Hepatology 7: 425-515); insulin gene controlregion which is active in pancreatic beta cells (Hanahan et al., 1985,Nature 315: 115-122), immunoglobulin gene control region which is activein lymphoid cells (Grosschedl et al., 1984, Cell 38: 647-658; Adams etal., 1985, Nature 318: 533-538; Alexander et al., 1987, Mol. Cell Biol.7: 1436-1444), mouse mammary tumor virus control region which is activein testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell45: 485-495), albumin gene control region which is active in liver(Pinckert et al., 1987, Genes and Devel. 1: 268-276), alpha-fetoproteingene control region which is active in liver (Krumlauf et al., 1985,Mol. Cell. Biol. 5: 1639-1648; Hammer et al., 1987, Science 235: 53-58),alpha-1 antitrypsin gene control region which is active in liver (Kelseyet al., 1987, Genes and Devel. 1: 161-171), beta globin gene controlregion which is active in myeloid cells (Mogram et al., 1985, Nature315: 338-340; Kollias et al., 1986, Cell 46: 89-94), myelin basicprotein gene control region which is active in oligodendrocyte cells ofthe brain (Readhead et al., 1987, Cell 48: 703-712), myosin lightchain-2 gene control region which is active in skeletal muscle (Sani1985, Nature 314: 283-286), and gonadotrophic releasing hormone genecontrol region which is active in gonadotrophs of the hypothalamus(Mason et al., 1986, Science 234: 1372-1378).

In a specific embodiment, a vector is used that comprises a promoteroperably linked to nucleic acid sequences encoding 53BP2 and/or a53BP2-IP (e.g. β-tubulin, p62, hnRNP G, 53BP2-IP1, 53BP2-IP2 or53BP2-IP3), or a fragment, derivative or homolog, thereof, one or moreorigins of replication, and optionally, one or more selectable markers(e.g., an antibiotic resistance gene). In a preferred embodiment, avector is used that comprises a promoter operably linked to nucleic acidsequences encoding both 53BP2 and a 53BP2-IP, one or more origins ofreplication, and optionally, one or more selectable markers.

In another specific embodiment, an expression vector containing thecoding sequences, or portions thereof, of 53BP2 and a 53BP2-IP (e.g.,β-tubulin, p62, hnRNP G, 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3), eithertogether or separately, is made by subcloning the gene sequences intothe EcoRI restriction site of each of the three PGEX vectors(glutathione S-transferase expression vectors; Smith and Johnson, 1988,Gene 7:31-40). This allows for the expression of products in the correctreading frame.

Expression vectors containing the sequences of interest can beidentified by three general approaches: (a) nucleic acid hybridization,(b) presence or absence of “marker” gene function, and (c) expression ofthe inserted sequences. In the first approach, 53BP2, β-tubulin, p62,hnRNP G, 53BP2-IP1, 53BP2-IP2, 53BP2-IP3, or other 53BP2-IP sequencescan be detected by nucleic acid hybridization to probes comprisingsequences homologous and complementary to the inserted sequences. In thesecond approach, the recombinant vector/host system can be identifiedand selected based upon the presence or absence of certain “marker”functions (e.g., binding to an anti-53BP2, anti-53BP2-IP, oranti-53BP2:53BP2-IP complex antibody, resistance to antibiotics,occlusion body formation in baculovirus, etc.) caused by insertion ofthe sequences of interest in the vector. For example, if 53BP2 or a53BP2-IP gene, or portion thereof, is inserted within the marker genesequence of the vector, recombinants containing the 53BP2 or 53BP2-IPfragment will be identified by the absence of the marker gene function.In the third approach, recombinant expression vectors can be identifiedby assaying for the 53BP2, β-tubulin, p62, hnRNP G, 53BP2-IP1,53BP2-IP2, 53BP2-IP3, or other 53BP2-IP products expressed by therecombinant vector. Such assays can be based, for example, on thephysical or functional properties of the interacting species in in vitroassay systems, e.g., formation of a 53BP2:53BP2-IP complex,immunoreactivity to antibodies specific for the protein.

Once recombinant 53BP2, β-tubulin, p62, hnRNP G, 53BP2-IP1, 53BP2-IP2,or 53BP2-IP3, or other 53BP2-IP molecules are identified and thecomplexes or individual proteins isolated, several methods known in theart can be used to propagate them. Once a suitable host system andgrowth conditions have been established, recombinant expression vectorscan be propagated and amplified in quantity. As previously described,the expression vectors or derivatives which can be used include, but arenot limited to: human or animal viruses such as vaccinia virus oradenovirus; insect viruses such as baculovirus, yeast vectors;bacteriophage vectors such as lambda phage; and plasmid and cosmidvectors.

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies or processes theexpressed proteins in the specific fashion desired. Expression fromcertain promoters can be elevated in the presence of certain inducers;thus expression of the genetically-engineered 53BP2 and/or 53BP2-IP maybe controlled. Furthermore, different host cells have characteristic andspecific mechanisms for the translational and post-translationalprocessing and modification (e.g. glycosylation, phosphorylation, etc.)of proteins. Appropriate cell lines or host systems can be chosen toensure the desired modification and processing of the foreign protein isachieved. For example, expression in a bacterial system can be used toproduce an unglycosylated core protein, while expression in mammaliancells ensures “native” glycosylation of a heterologous protein.Furthermore, different vector/host expression systems may effectprocessing reactions to different extents.

In other specific embodiments, the 53BP2 and/or 53BP2-IPs or fragments,homologs or derivatives thereof, may be expressed as fusion or chimericprotein products comprising the protein, fragment, homolog, orderivative joined via a peptide bond to a heterologous protein sequenceof a different protein. Such chimeric products can be made by ligatingthe appropriate nucleic acid sequences encoding the desired amino acidsto each other by methods known in the art, in the proper coding frame,and expressing the chimeric products in a suitable host by methodscommonly known in the art. Alternatively, such a chimeric product can bemade by protein synthetic techniques, e.g., by use of a peptidesynthesizer. Chimeric genes comprising portions of 53BP2 and/or a53BP2-IP, or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 fused to anyheterologous protein-encoding sequences may be constructed. A specificembodiment relates to a chimeric protein comprising a fragment of 53BP2and/or a 53BP2-IP, or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 of at least sixamino acids.

In a specific embodiment, fusion proteins are provided that contain theinteracting domains of the 53BP2 protein and a 53BP2-IP (e.g.,β-tubulin, p62, hnRNP G, 53BP2-IP1, 53BP2-IP2 or 53BP2-IP3) and,optionally, a peptide linker between the two domains, where such alinker promotes the interaction of the 53BP2 and 53BP2-IP bindingdomains. These fusion proteins may be particularly useful where thestability of the interaction is desirable (due to the formation of thecomplex as an intramolecular reaction), for example in production ofantibodies specific to the 53BP2:53BP2-IP complex.

In particular, 53BP2 and/or 53BP2-IP or 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 derivatives can be made by altering their sequences bysubstitutions, additions or deletions that provide for functionallyequivalent molecules. Due to the degeneracy of nucleotide codingsequences, other DNA sequences which encode substantially the same aminoacid sequence as a 53BP2 or 53BP2-IP or 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 gene can be used in the practice of the present invention.These include but are not limited to nucleotide sequences comprising allor portions of 53BP2, β-tubulin, p62, hnRNP G, 53BP2-IP1, 53BP2-IP2, or53BP2-IP3, or other 53BP2-IP genes that are altered by the substitutionof different codons that encode a functionally equivalent amino acidresidue within the sequence, thus producing a silent change. Likewise,the 53BP2, 53BP2-IP, 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 derivatives ofthe invention include, but are not limited to, those containing, as aprimary amino acid sequence, all or part of the amino acid sequence of53BP2 or a 53BP2-IP or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3, includingaltered sequences in which functionally equivalent amino acid residuesare substituted for residues within the sequence resulting in a silentchange. For example, one or more amino acid residues within the sequencecan be substituted by another amino acid of a similar polarity whichacts as a functional equivalent, resulting in a silent alteration.Substitutes for an amino acid within the sequence may be selected fromother members of the class to which the amino acid belongs. For example,the nonpolar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan and methionine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine. The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

In a specific embodiment of the invention, the nucleic acids encodingproteins and proteins consisting of or comprising a fragment of 53BP2 ora 53BP2-IP or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 consisting of at least6 (continuous) amino acids of 53BP2, a 53BP2-IP, 53BP2-IP1, 53BP2-IP2,or 53BP2-IP3 are provided. In other embodiments, the fragment consistsof at least 10, 20, 30, 40, or 50 amino acids of 53BP2, 53BP2-IP,53BP2-IP1, 53BP2-IP2, or 53BP2-IP3. In specific embodiments, suchfragments are not larger than 35, 100 or 200 amino acids. Derivatives oranalogs of 53BP2 and 53BP2-IPs or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3include but are not limited to molecules comprising regions that aresubstantially homologous to 53BP2, 53BP2-IPs, 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 in various embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%,90% or 95% identity over an amino acid sequence of identical size orwhen compared to an aligned sequence in which the alignment is done by acomputer homology program known in the art or whose encoding nucleicacid is capable of hybridizing to a sequence encoding 53BP2, a 53BP2-IP,53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 under stringent, moderatelystringent, or nonstringent conditions.

The 53BP2, 53BP2-IP, 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 derivatives andanalogs of the invention can be produced by various methods known in theart. The manipulations which result in their production can occur at thegene or protein level. For example, the cloned 53BP2, 53BP2-IP53BP2-IP1, or 53BP2-IP2 gene sequence can be modified by any of numerousstrategies known in the art (Maniatis, T., 1990, Molecular Cloning, ALaboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.). The sequences can be cleaved at appropriate sites withrestriction endonuclease(s), followed by further enzymatic modificationif desired, isolated, and ligated in vitro. In the production of thegene encoding a derivative or analog of 53BP2 or a 53BP2-IP care shouldbe taken to ensure that the modified gene retains the originaltranslational reading frame, uninterrupted by translational stopsignals, in the gene region where the desired activity is encoded.

Additionally, the 53BP2 and/or 53BP2-IP-encoding nucleic acid sequenceor 53BP2-IP1, 53BP2-IP2 or 53BP2-IP3-encoding nucleic acid sequence canbe mutated in vitro or in vivo, to create and/or destroy translation,initiation, and/or termination sequences, or to create variations incoding regions and/or form new restriction endonuclease sites or destroypre-existing ones, to facilitate further in vitro modification. Anytechnique for mutagenesis known in the art can be used, including butnot limited to, chemical mutagenesis and in vitro site-directedmutagenesis (Hutchinson et al., 1978, J. Biol. Chem 253:6551-6558), useof TAB® linkers (Pharmacia), etc.

Once a recombinant cell expressing 53BP2 and/or a 53BP2-IP or 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 protein, or fragment or derivative thereof, isidentified, the individual gene product or complex can be isolated andanalyzed. This is achieved by assays based on the physical and/orfunctional properties of the protein or complex, including, but notlimited to, radioactive labeling of the product followed by analysis bygel electrophoresis, immunoassay, cross-linking to marker-labeledproduct, etc.

The 53BP2:53BP2-IP complexes and 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3proteins may be isolated and purified by standard methods known in theart (either from natural sources or recombinant host cells expressingthe complexes or proteins), including but not restricted to columnchromatography (e.g., ion exchange, affinity, gel exclusion,reversed-phase high pressure, fast protein liquid, etc.), differentialcentrifugation, differential solubility, or by any other standardtechnique used for the purification of proteins. Functional propertiesmay be evaluated using any suitable assay known in the art.

Alternatively, once a 53BP2-IP or its derivative is identified, theamino acid sequence of the protein can be deduced from the nucleic acidsequence of the chimeric gene from which it was encoded. As a result,the protein or its derivative can be synthesized by standard chemicalmethods known in the art (e.g. see Hunkapiller et al, 1984, Nature 310:105-111).

In a specific embodiment of the present invention, such 53BP2:53BP2-IPcomplexes and 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 proteins, whetherproduced by recombinant DNA techniques, chemical synthesis methods, orby purification from native sources, include but are not limited tothose containing, as a primary amino acid sequence, all or part of theamino acid sequences substantially as depicted in FIGS. 1-4, and 10B, D,and F (SEQ ID NOS:2, 4, 6, 8, 13, 12 and 11), as well as fragments andother analogs and derivatives thereof, including proteins homologousthereto.

Manipulations of 53BP2 and/or 53BP2-IP sequences or 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 sequences may be made at the protein level.Included within the scope of the invention are complexes of 53BP2 or53BP2-IP fragments, derivatives or analogs and 53BP2-IP1, 53BP2-IP2, and53BP2-IP3 fragments, derivatives and analogs which are differentiallymodified during or after translation, e.g., by glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including but notlimited to specific chemical cleavage by cyanogen bromide, trypsin,chymotrypsin, papain, V8 protease, NaBH₄, acetylation, formylation,oxidation, reduction, metabolic synthesis in the presence oftunicamycin, etc.

In specific embodiments, the 53BP2 and/or 53BP2-IP sequences aremodified to include a fluorescent label. In another specific embodimentthe 53BP2 and/or the 53BP2-IP are modified to have a heterofunctionalreagent, such heterofunctional reagents can be used to crosslink themembers of the complex.

In addition, complexes of analogs and derivatives of 53BP2 and/or a53BP2-IP or analogs and derivatives of 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 can be chemically synthesized. For example, a peptidecorresponding to a portion of 53BP2 and/or a 53BP2-IP or 53BP2-IP1,53BP2-IP2 or 53BP2-IP3, which comprises the desired domain or whichmediates the desired activity in vitro (e.g., 53BP2:53BP2-IP complexformation), can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into the 53BP2and/or 53BP2-IP or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 sequence.Non-classical amino acids include but are not limited to the D-isomersof the common amino acids, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-aminobutyric acid, ε-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionoic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, fluoro-amino acids, designer amino acids such as β-methylamino acids, Ca-methyl amino acids, Na-methyl amino acids, and aminoacid analogs in general. Furthermore, the amino acid can be D(dextrorotary) or L (levorotary).

In cases where natural products are suspected of being mutant or areisolated from new species, the amino acid sequence of 53BP2, a 53BP2-IP,53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 isolated from the natural source, aswell as those expressed in vitro, or from synthesized expression vectorsin vivo or in vitro, can be determined from analysis of the DNAsequence, or alternatively, by direct sequencing of the isolatedprotein. Such analysis may be performed by manual sequencing or throughuse of an automated amino acid sequenator.

The 53BP2:53BP2-IP complexes and 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3proteins may also be analyzed by hydrophilicity analysis (Hopp andWoods, 1981, Proc. Natl. Acad. Sci. USA 78: 3824-3828). A hydrophilicityprofile can be used to identify the hydrophobic and hydrophilic regionsof the proteins, and help predict their orientation in designingsubstrates for experimental manipulation, such as in bindingexperiments, antibody synthesis, etc. Secondary structural analysis canalso be done to identify regions of the 53BP2 and/or a 53BP2-IP or53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 that assume specific structures (Chouand Fasman, 1974, Biochemistry 13: 222-23). Manipulation, translation,secondary structure prediction, hydrophilicity and hydrophobicityprofiles, open reading frame prediction and plotting, and determinationof sequence homologies, can be accomplished using computer softwareprograms available in the art.

Other methods of structural analysis including but not limited to X-raycrystallography (Engstrom, 1974 Biochem. Exp. Biol. 11:7-13), massspectroscopy and gas chromatography (Methods in Protein Science, J.Wiley and Sons, New York, 1997), and computer modeling (Fletterick andZoller, eds., 1986, Computer Graphics and Molecular Modeling, In:Current Communications in Molecular Biology, Cold Spring HarborLaboratory, Cold Spring Harbor Press, New York) can also be employed.

Identification and Isolation of 53BP2-IP1, 53BP2-IP2 and 53BP2-IP3 Genes

The invention relates to the nucleotide sequences of nucleic acidsencoding 53BP2-IP1, 53BP2-IP2 and 53BP2-IP3. In specific embodiments,the 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 nucleic acids comprise thesequence of SEQ ID NO:10, or the coding regions thereof, or nucleotidesequences encoding, in whole or in part, a 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 protein (e.g., a protein comprising the sequence of SEQ IDNO:11, 12, or 13, respectively). The invention provides purified nucleicacids consisting of at least 8 nucleotides (i.e., a hybridizableportion) of a 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 sequence; in otherembodiments, the nucleic acids consist of at least 25 (continuous)nucleotides, 50 nucleotides, 100 nucleotides, 150 nucleotides, or 200nucleotides of a 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 sequence, or afull-length 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 coding sequence. Inanother embodiment, the nucleic acids are smaller than 35, 200 or 500nucleotides in length. Nucleic acids can be single or double stranded.The invention also relates to nucleic acids hybridizable to orcomplementary to the foregoing sequences, in particular the inventionprovides the inverse complement to nucleic acids hybridizable to theforegoing sequences (i.e. the inverse complement of a nucleic acidstrand has the complementary sequence running in reverse orientation tothe strand so that the inverse complement would hybridize withoutmismatches to the nucleic acid strand; thus, for example, where thecoding strand is hybridizable to a nucleic acid with no mismatchesbetween the coding strand and the hybridizable strand, then the inversecomplement of the hybridizable strand is identical to the codingstrand). In specific aspects, nucleic acids are provided which comprisea sequence complementary to (specifically are the inverse complement of)at least 10, 25, 50, 100, or 200 nucleotides or the entire coding regionof a 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 gene.

In a specific embodiment, a nucleic acid which is hybridizable to a53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 nucleic acid (e.g., having sequenceSEQ ID NO:10), or to a nucleic acid encoding a 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 derivative, under conditions of low stringency is provided. Byway of example and not limitation, procedures using such conditions oflow stringency are as follows (see also Shilo and Weinberg, 1981, Proc.Natl. Acad. Sci. USA 78:6789-6792): Filters containing DNA arepretreated for 6 hours at 40° C. in a solution containing 35% formamide,5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1%BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations arecarried out in-the same solution with the following modifications: 0.02%PVP, 0.02% Ficoll, 0.2% BSA, 100 gg/ml salmon sperm DNA, 10% (wt/vol)dextran sulfate, and 5-20×10⁶ cpm ³²P-labeled probe is used. Filters areincubated in hybridization mixture for 18-20 hours at 40° C., and thenwashed for 1.5 hours at 55° C. in a solution containing 2×SSC, 25 mMTris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution isreplaced with fresh solution and incubated an additional 1.5 hours at60° C. Filters are blotted dry and exposed for autoradiography. Ifnecessary, filters are washed for a third time at 65-68° C. andreexposed to film. Other conditions of low stringency which may be usedare well known in the art (e.g., as employed for cross-specieshybridizations).

In another specific embodiment, a nucleic acid which is hybridizable toa 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 nucleic acid under conditions ofhigh stringency is provided. By way of example and not limitation,procedures using such conditions of high stringency are as follows:Prehybridization of filters containing DNA is carried out for 8 hours toovernight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/mldenatured salmon sperm DNA. Filters are hybridized for 48 hours at 65°C. in prehybridization mixture containing 100 μg/ml denatured salmonsperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe. Washing of filters isdone at 37° C. for 1 hour in a solution containing 2×SSC, 0.01% PVP,0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1×SSC at50° C. for 45 minutes before autoradiography. Other conditions of highstringency which may be used are well known in the art.

In another specific embodiment, a nucleic acid, which is hybridizable toa 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 nucleic acid under conditions ofmoderate stringency is provided. For example, but not limited to,procedures using such conditions of moderate stringency are as follows:Filters containing DNA are pretreated for 6 hours at 55° C. in asolution containing 6×SSC, 5×Denhart's solution, 0.5% SDS and 100 μg/mldenatured salmon sperm DNA. Hybridizations are carried out in the samesolution and 5-20×10⁶ cpm ³²P-labeled probe is used. Filters areincubated in hybridization mixture for 18-20 hours at 55° C., and thenwashed twice for 30 minutes at 60° C. in a solution containing 1×SSC and0.1% SDS. Filters are blotted dry and exposed for autoradiography. Otherconditions of moderate stringency which may be used are well-known inthe art. Washing of filters is done at 37° C. for 1 hour in a solutioncontaining 2×SSC, 0.1% SDS.

Nucleic acids encoding derivatives and analogs of 53BP2-IP1, 53BP2-IP2,or 53BP2-IP3 proteins (see Section 5.2), and 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 antisense nucleic acids (see Section 5.5.7) are additionallyprovided. As is readily apparent, as used herein, a “nucleic acidencoding a fragment or portion of a 53BP2-IP1, 53BP2-IP2 or 53BP2-IP3”shall be construed as referring to a nucleic acid encoding only therecited fragment or portion of 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3protein, and not the other contiguous portions of the 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 as a continuous sequence.

Fragments of 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 nucleic acids comprisingregions conserved between (with homology to) other 53BP2-IP1, 53BP2-IP2,or 53BP2-IP3 nucleic acids, of the same or different species, are alsoprovided.

Nucleic acids predicted to encode (at least in part) 53BP2-IP1,53BP2-IP2, and 53BP2-IP3 were identified as encoding a protein orproteins that interact with 53BP2 using the improved version yeast twohybrid system (e.g. as described in Section 5.7.1 and exemplified inSection 6.1 supra). The present inventors found that the 5′ end of thisidentified nucleic acid (illustrated in FIG. 9) has a nucleotidesequence identical to the nucleotide sequence of the EST sequence ESTR72810.

EST sequences are part of human DNA databases such as the GenBankdatabase “dbest”. These sequences typically represent incompletefragments of putative genes not yet ascribed to encode a known proteinor RNA species. These sequences generally do not encode a full-lengthprotein because they generally (1) lack a methionine codon to act as asite of translational initiation; (2) lack a translational stop codon;or (3) do not contain an open reading frame to code for a protein longerthan approximately 60 amino acids (shorter than the smallest knowntranslated protein). The EST databases contain many overlappingsequences. Thus, it is possible to find contiguous sequences to assemblea longer sequence representative of a larger original sequence found innature. Common in silico procedures known in the art, including use ofthe “BLAST” family of programs available through the National Center forBiotechnology Information (N.C.B.I.), can be used to detect homologiesbetween nucleic acid sequences in the databases. To account forsequencing errors, silent mutations, etc., present in the database, asignificant homology can be defined as 100%, 99%, 98%, 97%, 96%, 95% orsome lesser level over a span of 20, 25, 30, 35, 40 or greater span ofnucleotide overlap. The homology detection paradigm may allow for alimited number of single or, at most, double nucleotide insertion ordeletion mismatches, particularly in regions of sequences known to bedifficult to sequence, such as very high GC regions, multiple Gresidues, etc.

These in silico procedures allow for the “assembly” of two sequencesthat overlap nonidentical spans of a common sequence. This assembledsequence, in turn, is used to identify further related sequences by thesame procedure. The 5′ and 3′ ends of the assembled sequence areextended until significant homology to sequences within availabledatabases cannot be detected. The assembled EST sequence can besubjected to a final search of available databases to detect homologiesto known protein sequences that were not detected over the shorter spanof the original EST sequence.

The present inventors identified several EST sequences that overlap withEST R72810 at both the 5′ and 3′ termini. These sequences were assembledas described in Section 6.3 infra and as depicted in FIGS. 8 and 9.

The assembled EST sequence can be analyzed by a number of nucleic acidanalysis programs available in the art to define possible proteintranslation products of the assembled nucleic acid sequence. Translationin all six phases will define possible open reading frames (contiguousspans of codons for amino acids without the presence of a stop codon).In the case where EST sequences are derived from directionally clonedlibraries, only the three forward (5′ to 3′) translations are requiredbecause the sense, or coding strand, of the EST is already defined. Thepresence of ATG codons that define possible sites of initiation ofprotein translation identify the beginning of such an open readingframe. If an open reading frame extends to the 5′ end of the assemblednucleic acid sequence is longer than 60 amino acid residues, theassembled EST sequence encodes a potential C-terminus of a proteinwithin that reading frame, i.e., a protein that is missing one or moreN-terminal amino acids.

In silico analysis of the assembled sequence revealed three possibletranslation products, 53BP2-IP1 (for 53BP2-Interacting Protein 1),53BP2-IP2 (for 53BP2-Interacting Protein 2), and 53BP2-IP3 (for53BP2-Interacting Protein 3).

Any method available in the art can be used to obtain a full length(i.e., encompassing the entire coding region) cDNA or genomic DNA cloneencoding 53BP2-IP1 and/or 53BP2-IP2 and/or 53BP2-IP3. In particular, thepolymerase chain reaction (PCR) can be used to amplify the sequenceassembled from the EST sequences in a genomic or cDNA library.Oligonucleotide primers that hybridize to sequences at the 3′ and 5′termini of the assembled EST sequences can be used as primers to amplifyby PCR sequences from a nucleic acid sample (RNA or DNA), preferably acDNA library, from an appropriate source (e.g. the sample from which theinitial CDNA library for the yeast two hybrid assay fusion populationwas derived).

PCR can be carried out., e.g., by use of a Perkin-Elmer Cetus thermalcycler and Taq polymerase (Gene Amp™). The DNA being amplified caninclude mRNA or cDNA or genomic DNA from any eukaryotic species. One canchoose to synthesize several different degenerate primers, for use inthe PCR reactions. It is also possible to vary the stringency ofhybridization conditions used in priming the PCR reactions, to amplifynucleic acid homologs (e.g., to obtain 53BP2-IP1, 53BP2-IP2 and53BP2-IP3 sequences from species other than humans or to obtain humansequences with homology to 53BP2-IP1 and/or 53BP2-IP2 and/or 53BP2-IP3)by allowing for greater or lesser degrees of nucleotide sequencesimilarity between the known nucleotide sequence and the nucleic acidhomolog being isolated. For cross species hybridization, low stringencyconditions are preferred. For same species hybridization, moderatelystringent conditions are preferred.

After successful amplification of the nucleic acid containing all or aportion of the sequence assembled from the EST sequences or of a nucleicacid encoding all or a portion of a 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3homolog, that segment may be molecularly cloned and sequenced, andutilized as a probe to isolate a complete cDNA or genomic clone. This,in turn, will permit the determination of the gene's complete nucleotidesequence, the analysis of its expression, and the production of itsprotein product for functional analysis, as described infra. In thisfashion, the nucleotide sequences of the entire 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 genes as well as additional genes encoding 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 proteins and analogs may be identified.

Any eukaryotic cell potentially can serve as the nucleic acid source forthe molecular cloning of the 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 gene.The nucleic acids can be isolated from vertebrate, mammalian, human,porcine, bovine, feline, avian, equine, canine, as well as additionalprimate sources, insects, plants, etc. The DNA may be obtained bystandard procedures known in the art from cloned DNA (e.g., a DNA“library”), by chemical synthesis, by cDNA cloning, or by the cloning ofgenomic DNA, or fragments thereof, purified from the desired cell (see,for example, Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; Glover, D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRLPress, Ltd., Oxford, U.K. Vol. I, II). Clones derived from genomic DNAmay contain regulatory and intron DNA regions in addition to codingregions; clones derived from cDNA will contain only exon sequences.Whatever the source, the gene should be molecularly cloned into asuitable vector for propagation of the gene.

In the molecular cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will encode the desired gene. The DNA may becleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, for example, bysonication. The linear DNA fragments can then be separated according tosize by standard techniques, including but not limited to, agarose andpolyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired gene may be accomplished in a number ofways. For example, a portion of the 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3(of any species) gene (e.g., a PCR amplification product obtained asdescribed above or an oligonucleotide having a sequence of a portion ofthe known nucleotide sequence) or its specific RNA, or a fragmentthereof be purified and labeled, and the generated DNA fragments may bescreened by nucleic acid hybridization to the labeled probe (Benton, W.and Davis, R., 1977, Science 196:180; Grunstein, M. And Hogness, D.,1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). Those DNA fragments withsubstantial homology to the probe will hybridize. It is also possible toidentify the appropriate fragment by restriction enzyme digestion(s) andcomparison of fragment sizes with those expected according to a knownrestriction map if such is available or by DNA sequence analysis andcomparison to the known nucleotide sequence of53BP2-IP1/53BP2-IP2/53BP2-IP3. Further selection can be carried out onthe basis of the properties of the gene. Alternatively, the presence ofthe gene may be detected by assays based on the physical, chemical, orimmunological properties of its expressed product. For example, cDNAclones, or DNA clones which hybrid-select the proper mRNAs, can beselected which produce a protein that, e.g., has similar or identicalelectrophoretic migration, isolectric focusing behavior, proteolyticdigestion maps, or antigenic properties or ability to bind 53BP2, asknown for 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3. If an anti-53BP2-IP1,anti-53BP2-IP2 or anti-53BP2-IP3 antibody is available, the protein maybe identified by binding of labeled antibody to the putatively53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 synthesizing clones, in an ELISA(enzyme-linked immunosorbent assay)-type procedure.

Alternatives to isolating the 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 genomicDNA include, but are not limited to, chemically synthesizing the genesequence itself from a known sequence or making cDNA to the mRNA thatencodes the 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 protein. For example, RNAfor cDNA cloning of the 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 gene can beisolated from cells expressing the protein. Other methods are possibleand within the scope of the invention.

The identified and isolated nucleic acids can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas PBR322 or pUC plasmid derivatives or the Bluescript vector(Stratagene, La Jolla, Calif.). The insertion into a cloning vector can,for example, be accomplished by ligating the DNA fragment into a cloningvector which has complementary cohesive termini. However, if thecomplementary restriction sites used to fragment the DNA are not presentin the cloning vector, the ends of the DNA molecules may beenzymatically modified. Alternatively, any site desired may be producedby ligating nucleotide sequences (linkers) onto the DNA termini; theseligated linkers may comprise specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences. In an alternative method, the cleaved vector and 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 gene may be modified by homopolymeric tailing.Recombinant molecules can be introduced into host cells viatransformation, transfection, infection, electroporation, etc., so thatmany copies of the gene sequence are generated.

In an alternative method, the desired gene may be identified andisolated after insertion into a suitable cloning vector in a “shot gun”approach. Enrichment for the desired gene, for example, by sizefractionization, can be done before insertion into the cloning vector.

In specific embodiments, transformation of host cells with recombinantDNA molecules that incorporate the isolated 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 gene, cDNA, or synthesized DNA sequence enables generation ofmultiple copies of the gene. Thus, the gene may be obtained in largequantities by growing transformants, isolating the recombinant DNAmolecules from the transformants and, when necessary, retrieving theinserted gene from the isolated recombinant DNA.

The 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 sequences provided by the instantinvention include those nucleotide sequences encoding substantially thesame amino acid sequences as found in native 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 proteins, and those encoded amino acid sequences withfunctionally equivalent amino acids, as well as those encoding other53BP2-IP1, 53BP2-IP2 or 53BP2-IP3 derivatives or analogs, as describedin Section 5.1 supra for 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 derivativesand analogs.

Antibodies to 53BP2:53BP2-IP Complexes, 53BP2-IP1, 53BP2-IP2, and53BP2-IP3 Protiens

According to the invention, the 53BP2:53BP2-IP complexes (e.g. 53BP2-IPcomplexes with β-tubulin, p62, hnRNP G, 53BP2-IP1, 53BP2-IP2, or53BP2-IP3), or fragments, derivatives or homologs thereof, or 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 protein and fragments, homologs and derivativesthereof may be used as immunogens to generate antibodies whichimmunospecifically bind such immunogens. Such antibodies include but arenot limited to polyclonal, monoclonal, chimeric, single chain, Fabfragments, and an Fab expression library. In a specific embodiment,antibodies to complexes of human 53BP2 and human 53BP2-IPs are produced.In another embodiment, complexes formed from fragments of 53BP2 and a53BP2-IP, which fragments contain the protein domain that interacts withthe other member of the complex, are used as immunogens for antibodyproduction. In another specific embodiment, 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 or fragments, derivatives, or homologs thereof are used asimmunogens.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to a 53BP2:53BP2-IP complex, derivative or analog,or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 protein, derivatives, fragments oranalogs.

For production of the antibody, various host animals can be immunized byinjection with the native 53BP2:53BP2-IP complex, or 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 protein or a synthetic version, or a derivativeof the foregoing, such as a cross-linked 53BP2:53BP2-IP, such hostanimals include but are not limited to rabbits, mice, rats, etc. Variousadjuvants can be used to increase the immunological response, dependingon the host species, and include but are not limited to Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, dinitrophenol, and potentiallyuseful human adjuvants such as bacille Calmette-Guerin (BCG) andcorynebacterium parvum.

For preparation of monoclonal antibodies directed towards a53BP2:53BP2-IP complex or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 orderivatives, fragments or analogs thereof, any technique that providesfor the production of antibody molecules by continuous cell lines inculture may be used. Such techniques include but are not restricted tothe hybridoma technique originally developed by Kohler and Milstein(1975, Nature 256: 495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4: 72), andthe EBV hybridoma technique to produce human monoclonal antibodies (Coleet al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96). In an additional embodiment of the invention,monoclonal antibodies can be produced in germ-free animals utilizingrecent technology (PCT/US90/02545). According to the invention, humanantibodies may be used and can be obtained by using human hybridomas(Cote et al., 1983, Proc. Natl. Acad. Sci. USA 80: 2026-2030). Or bytransforming human B cells with EBV virus in vitro (Cole et al., 1985,in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). In fact, according to the invention, techniques developed forthe production of “chimeric antibodies” (Morrison et al., 1984, Proc.Natl. Acad. Sci. USA 81: 6851-6855; Neuberger et al., 1984, Nature312:604-608; Takeda et al., 1985, Nature 314: 452-454) by splicing thegenes from a mouse antibody molecule specific for the 53BP2:53BP2complex or 53BP2-IP1, or 53BP2-IP2 protein together with genes from ahuman antibody molecule of appropriate biological activity can be used;such antibodies are within the scope of this invention.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce 53BP2:53BP2-IP complex-specific and 53BP2-IP1, 53BP2-IP2, and53BP2-IP3-specific single chain antibodies. An additional embodiment ofthe invention utilizes the techniques described for the construction ofFab expression libraries (Huse et al., 1989, Science 246: 1275-1281) toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity for 53BP2: β-tubulin, p62, hnRNP G, 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 complexes, or 53BP2-IP1, 53BP2-IP2 or 53BP2-IP3derivatives, or analogs thereof. Non-human antibodies can be “humanized”by known methods (see, e.g., U.S. Pat. No. 5,225,539).

Antibody fragments that contain the idiotypes of 53BP2:53BP2-IPcomplexes or of 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 can be generated bytechniques known in the art. For example, such fragments include but arenot limited to: the F(ab′)2 fragment which can be produced by pepsindigestion of the antibody molecule; the Fab′ fragments that can begenerated by reducing the disulfide bridges of the F(ab′)2 fragment, theFab fragments that can be generated by treating the antibody molecularwith papain and a reducing agent, and Fv fragments.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., ELISA(enzyme-linked immunosorbent assay). To select antibodies specific to aparticular domain of the 53BP2:53BP2 complex or 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 one may assay generated hybridomas for a product that binds tothe fragment of the 53BP2:53BP2-IP complex or 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 that contains such a domain. For selection of an antibody thatspecifically binds a 53BP2:53BP2-IP complex but which does notspecifically bind to the individual proteins of the 53BP2-IP complex,one can select on the basis of positive binding to the 53BP2:53BP2-IPcomplex and a lack of binding to the individual 53BP2 and 53BP2-IPproteins.

Antibodies specific to a domain of the 53BP2:53BP2-IP complex are alsoprovided, as are antibodies to specific domains of 53BP2-IP1, 53BP2-IP2,and 53BP2-IP3.

The foregoing antibodies can be used in methods known in the artrelating to the localization and/or quantitation of 53BP2:53BP2-IPcomplexes and 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 proteins of theinvention, e.g., for imaging these proteins, measuring levels thereof inappropriate physiological samples, in diagnostic methods, etc.

In another embodiment of the invention (see infra), anti-53BP2:53BP2-IPcomplex antibodies and fragments thereof, or anti-53BP2-IP1,anti-53BP2-IP2, and anti-53BP2-IP3 or fragments thereof, containing thebinding domain, are Therapeutics.

Diagnostic, Prognostic, and Screening Uses of 53BP2:53BP-IP Complexesand 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 Protiens

53BP2:53BP2-IP complexes (particularly 53BP2 complexed with β-tubulin,p62, hnRNP G, 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3), or 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 proteins, may be markers of specific diseasestates involving disruption of cell cycle progression, cellularapoptosis and/or differentiation, intracellular signal transduction,protein transport, and/or c-Src activation, transcriptional ortranslational regulation, tumorigenesis and tumor progression,ubiquitin-mediated proteolysis, mRNA binding and metabolism, and effectson autoimmune processes, and thus have diagnostic utility. Further,definition of particular groups of patients with elevations ordeficiencies of a 53BP2:53BP-IP complex or 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 protein can lead to new nosological classifications ofdiseases, furthering diagnostic ability.

Detecting levels of 53BP2:53BP-IP complexes, or individual proteins thathave been shown to form complexes with 53BP2, or the 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 proteins, or detecting levels of the mRNAencoding the components of the 53BP2-IP:53BP2-IP complexes, or the53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 proteins may be used in prognosis, tofollow the course of disease states, to follow therapeutic response,etc. 53BP2:53BP2-IP complexes and the individual components of the53BP2:53BP2-IP complexes (e.g., 53BP2, β-tubulin, p62, hnRNP G,53BP2-IP1, 53BP2-IP2, and 53BP2-IP3), and derivatives, analogs andsubsequences thereof, 53BP2 and/or 53BP2-IP, 53BP2-IP1, 53BP2-IP2, and53BP2-IP3 nucleic acids (and sequences complementary thereto), andanti-53BP2:53BP2-IP complex antibodies and antibodies directed againstthe individual components that can form 53BP2:53BP2-IP complexes andanti-53BP2-IP1, anti-53BP2-IP2, and anti-53BP2-IP3 antibodies, have usesin diagnostics. Such molecules can be used in assays, such asimmunoassays, to detect, prognose, diagnose, or monitor variousconditions, diseases, and disorders characterized by aberrant levels of53BP2:53BP2-IP complexes or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 proteins,or monitor the treatment thereof. In a specific embodiment, a53BP2:53BP2-IP complex is detected that is not a complex of 53BP2 andPP1 or a complex of 53BP2 and p53.

In particular, such an immunoassay is carried out by a method comprisingcontacting a sample derived from a patient with an anti-53BP2:53BP2-IPcomplex antibody or anti-53BP2-IP1, anti-53BP2-IP2, or anti-53BP2-IP3antibody under conditions such that immunospecific binding can occur,and detecting or measuring the amount of any immunospecific binding bythe antibody. In a specific aspect, such binding of antibody, in tissuesections, can be used to detect aberrant 53BP2:53BP2-IP complex or53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 protein localization or aberrant(e.g., high, low or absent) levels of 53BP2:53BP2-IP complex orcomplexes or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3. In a specificembodiment, antibody to 53BP2:53BP2-IP complex can be used to assay in apatient tissue or serum sample for the presence of 53BP2:53BP2-IPcomplex where an aberrant level of 53BP2:53BP2-IP complex is anindication of a diseased condition. In another embodiment, antibody to53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 can be used to assay in a patienttissue or serum sample for the presence of 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 where an aberrant level of 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3is an indication of a diseased condition. By “aberrant levels,” is meantincreased or decreased levels relative to that present, or a standardlevel representing that present, in an analogous sample from a portionof the body or from a subject not having the disorder.

The immunoassays which can be used include but are not limited tocompetitive and non-competitive assay systems using techniques such asWestern blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement-fixation assays, immunoradiometricassays, fluorescent immunoassays, protein A immunoassays, to name but afew.

Nucleic acids encoding the components of the 53BP2:53BP2-IP complexes(e.g., 53BP2, β-tubulin, p62, hnRNP G, 53BP2-IP1, 53BP2-IP2, and53BP2-IP3) and the 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 proteins andrelated nucleic acid sequences and subsequences, including complementarysequences, can also be used in hybridization assays. The 53BP2,53BP2-IP, 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 nucleic acid sequences, orsubsequences thereof comprising about at least 8 nucleotides, can beused as hybridization probes. Hybridization assays can be used todetect, prognose, diagnose, or monitor conditions, disorders, or diseasestates associated with aberrant levels of the mRNAs encoding thecomponents of a 53BP2:53BP2-IP complex or 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 protein as described supra. In particular, such ahybridization assay is carried out by a method comprising contacting asample containing nucleic acid with a nucleic acid probe capable ofhybridizing to 53BP2, a 53BP2-IP, or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3DNA or RNA (including cDNA prepared from mRNA from a sample), underconditions such that hybridization can occur, and detecting or measuringany resulting hybridization. In a preferred aspect, the hybridizationassay is carried out using nucleic acid probes capable of hybridizing to53BP2 and to a binding partner of 53BP2 (e.g. β-tubulin, p62, hnRNP G,53BP2-IP1, 53BP2-IP2, or 53BP2-IP3) to measure concurrently theexpression of both members of a 53BP2:53BP2-IP complex.

In specific embodiments, diseases and disorders involving orcharacterized by aberrant levels of 53BP2:53BP2-IP complexes (e.g.,complexes of 53BP2 with β-tubulin, p62, hnRNP G, 53BP2-IP1, 53BP2-IP2,or 53BP2-IP3) can be diagnosed, or their suspected presence can bescreened for, or a predisposition to develop such disorders can bedetected, by detecting aberrant levels of 53BP2:53BP2-IP complexes, oruncomplexed 53BP2 and/or 53BP2-IP (e.g. β-tubulin, p62, hnRNP G,53BP2-IP1, 53BP2-IP2, or 53BP2-IP3) proteins or nucleic acids orfunctional activity including but not restricted to binding to aninteracting partner (e.g. 53BP2, β-tubulin, p62, hnRNP G, 53BP2-IP1,53BP2-IP2, or 53BP2-IP3), or by detecting mutations in 53BP2 and/or a53BP2-IP RNA, DNA or protein (e.g., translocations, truncations, changesin nucleotide or amino acid sequence relative to wild-type 53BP2 and/orthe 53BP2-IP) that cause increased or decreased expression or activityof a 53BP2:53BP2-IP complex and/or 53BP2 and/or a protein that binds to53BP2. Such diseases and disorders include but are not limited to thosedescribed in Section 5.5 and its subsections.

By way of example, levels of 53BP2:53BP2-IP complexes and the individualcomponents of 53BP2:53BP2-IP components can be detected by immunoassay,levels of 53BP2 and/or 53BP2-IP RNA can be detected by hybridizationassays (e.g., Northern blots, dot blots), binding of 53BP2 to a 53BP2-IPcan be done by binding assays commonly known in the art, translocationsand point mutations in 53BP2 and/or in genes encoding 53BP2-IPs can bedetected by Southern blotting, RFLP analysis, PCR using primers thatpreferably generate a fragment spanning at least most of the 53BP2and/or 53BP2-IP gene, sequencing of the 53BP2 and/or 53BP2-IP genomicDNA or cDNA obtained from the patient, etc.

Assays well known in the art (e.g. assays described above such asimmunoassays, nucleic acid hybridization assays, activity assays, etc.)can be used to determine whether one or more particular 53BP2:53BP2-IPcomplexes are present at either increased or decreased levels or areabsent in samples from patients suffering from a particular disease ordisorder or having a predisposition to develop such a disease ordisorder as compared to the levels in samples from subjects not havingsuch a disease or disorder. Additionally, these assays can be used todetermine whether the ratio of the 53BP2:53BP2-IP complex to theuncomplexed components of the 53BP2:53BP2-IP complex, i.e. 53BP2 and/orthe specific 53BP2-IP in the complex of interest (e.g., β-tubulin, p62,hnRNP G, 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3) is increased or decreasedin samples from patients suffering from a particular disease or disorderor having a predisposition to develop such a disease or disorder ascompared to the ratio in samples from subjects not having such a diseaseor disorder. In the event that levels of one or more particular53BP2:53BP2-IP complexes are determined to be increased in patientssuffering from a particular disease or disorder or having apredisposition to develop such a disease or disorder, then theparticular disease or disorder or predisposition for a disease ordisorder can be diagnosed, prognosed, screened for, or monitored bydetecting increased levels of the one or more 53BP2:53BP2-IP complexes,the mRNA that encodes the members of the one or more particular53BP2:53BP2-IP complexes (including detecting the corresponding in cDNAprepared from an mRNA sample), or 53BP2:53BP2-IP complex functionalactivity.

Accordingly, in a specific embodiment of the invention, diseases anddisorders involving increased levels of one or more 53BP2:53BP2-IPcomplexes can be diagnosed, or their suspected presence can be screenedfor, or a predisposition to develop such disorders can be detected, bydetecting increased levels of the one or more 53BP2:53BP2-IP complexes,the mRNA encoding both members of the complex, or complex functionalactivity, or by detecting mutations in 53BP2 or the 53BP2-IP (e.g.,translocations in nucleic acids, truncations in the gene or protein,changes in nucleotide or amino acid sequence relative to wild-type 53BP2or the 53BP2-IP) that inhibit 53BP2:53BP2-IP complex formation.

In the event that levels of one or more particular 53BP2:53BP2-IPcomplexes are determined to be decreased in patients suffering from aparticular disease or disorder or having a predisposition to developsuch a disease or disorder, then the particular disease or disorder orpredisposition for a disease or disorder can be diagnosed, prognosed,screened for, or monitored by detecting decreased levels of the one ormore 53BP2:53BP2-IP complexes, the mRNA that encodes the members of theparticular one or more 53BP2:53BP2-IP complexes, or 53BP2:53BP2-IPcomplex functional activity.

Accordingly, in a specific embodiment of the invention, diseases anddisorders involving decreased levels of one or more 53BP2:53BP2-IPcomplexes can be diagnosed, or their suspected presence can be screenedfor, or a predisposition to develop such disorders can be detected, bydetecting decreased levels of the one or more 53BP2:53BP2-IP complexes,the mRNA encoding the members of the one or more complexes, or complexfunctional activity, or by detecting mutations in 53BP2 or the 53BP2-IP(e.g., translocations in nucleic acids, truncations in the gene orprotein, changes in nucleotide or amino acid sequence relative towild-type 53BP2 or the 53BP2-IP) that stabilize or enhance53BP2:53BP2-IP complex formation.

In another specific embodiment, diseases and disorders involvingaberrant expression of 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 are diagnosed,or their suspected presence can be screened for, or a predisposition todevelop such disorders can be detected, by detecting aberrant levels of53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 protein, RNA, or functional activity,or by detecting mutations in 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 RNA, DNAor protein (e.g., translocations in nucleic acids, truncations in thegene or protein, changes in nucleotide or amino acid sequence relativeto wild-type 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3) that cause aberrantexpression or activity of 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3. Suchdiseases and disorders include but are not limited to those describedinfra Section 5.5. By way of example, levels of 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 RNA and protein, 53BP2 binding activity, and the presence oftranslocations or point mutations, can be determined as described above.

Assays well known in the art (e.g. assays described above such asimmunoassays, nucleic acid hybridization assays, activity assays, etc.)can be used to determine whether 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 ispresent at either increased or decreased levels or is absent in samplesfrom patients suffering from a particular disease or disorder or havinga predisposition to develop such a disease or disorder as compared tothe levels in samples from subjects not having such a disease ordisorder. In the event that levels of 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3are determined to be increased in patients suffering from a particulardisease or disorder or having a predisposition to develop such a diseaseor disorder, then the particular disease or disorder or predispositionfor a disease or disorder can be diagnosed, prognosed, screened for, ormonitored by detecting increased levels of 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 protein or mRNA, or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3functional activity (e.g. binding to 53BP2).

Accordingly, in a specific embodiment of the invention, diseases anddisorders involving increased levels of 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 can be diagnosed, or their suspected presence can be screenedfor, or a predisposition to develop such disorders can be detected, bydetecting increased levels of 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3proteins or nucleic acids of 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3functional activity, or by detecting mutations in 53BP2-IP1, 53BP2-IP2,or 53BP2-IP3 (e.g., translocations in nucleic acids, truncations in thegene or protein, changes in nucleotide or amino acid sequence relativeto wild-type 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3) that inhibit 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 functional activity.

In the event that levels of 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 aredetermined to be decreased in patients suffering from a particulardisease or disorder or having a predisposition to develop such a diseaseor disorder, then the particular disease or disorder or predispositionfor a disease or disorder can be diagnosed, prognosed, screened for, ormonitored by detecting decreased levels of the 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 proteins or nucleic acids, or 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 functional activity.

Accordingly, in a specific embodiment of the invention, diseases anddisorders involving decreased levels of 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 can be diagnosed, or their suspected presence can be screenedfor, or a predisposition to develop such disorders can be detected, bydetecting decreased levels of 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3, themRNA encoding 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 (including detectingthe corresponding cDNA of mRNA in cDNA prepared from an mRNA sample), orcomplex functional activity, or by detecting mutations in 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 (e.g., translocations in nucleic acids,truncations in the gene or protein, changes in nucleotide or amino acidsequence relative to wild-type 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3) thatstabilize or enhance 53BP2:53BP2-IP complex formation.

The use of detection techniques, especially those involving antibodiesagainst the 53BP2:53BP2-IP complexes or 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 proteins, provides a method of detecting specific cells thatexpress the complex or protein. Using such assays, specific cell typescan be defined in which one or more particular 53BP2:53BP2-IP complexes,or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 protein are expressed, and thepresence of the complex or protein can be correlated with cellviability.

Also embodied are methods to detect a 53BP2:53BP2-IP complex, or53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 protein, in cell culture models thatexpress particular 53BP2:53BP2-IP complexes or 53BP2-IP1, 53BP2-IP1, or53BP2-IP3 or derivatives thereof, for the purpose of characterizing orpreparing 53BP2:53BP2-IP complexes, or 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 for harvest. This embodiment includes cell sorting ofprokaryotes such as but not restricted to bacteria (Davey and Kell,1996, Microbiol. Rev. 60: 641-696), primary cultures and tissuespecimens from eukaryotes, including mammalian species such as human(Steele et al., 1996, Clin. Obstet. Gynecol 39: 801-813), and continuouscell cultures (Orfao and Ruiz-Arguelles, 1996, Clin. Biochem. 29: 5-9).

Kits for diagnostic use are also provided, that comprise in one or morecontainers an anti-53BP2:53BP2-IP complex antibody or an anti-53BP2-IP1,anti-53BP2-IP2, or anti-53BP2-IP3 antibody, and, optionally, a labeledbinding partner to the antibody. Alternatively, the anti-53BP2:53BP2-IPcomplex antibody or anti-53BP2-IP1, anti-53BP2-IP2, or anti-53BP2-IP3antibody can be labeled (with a detectable marker, e.g., achemiluminescent, enzymatic, fluorescent, or radioactive moiety). A kitis also provided that comprises in one or more containers a nucleic acidprobe capable of hybridizing to 53BP2 and/or a 53BP2-IP (e.g.,β-tubulin, p62, hnRNP G, 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3) RNA. In aspecific embodiment, a kit can comprise in one or more containers a pairof primers (e.g., each in the size range of 6-30 nucleotides) that arecapable of priming amplification [e.g., by polymerase chain reaction(see e.g., Innis et al., 1990, PCR Protocols, Academic Press, Inc., SanDiego, Calif.), ligase chain reaction (see EP 320,308) use of Qβreplicase, cyclic probe reaction, or other methods known in the art),under appropriate reaction conditions of at least a portion of a 53BP2and/or a 53BP2-IP or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 nucleic acid. Akit can optionally further comprise in a container a predeterminedamount of a purified 53BP2:53BP2-IP complex, 53BP2 and/or a 53BP2-IP or53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 or nucleic acids thereof, e.g., foruse as a standard or control.

Therapeutic Uses of 53BP2:53BP2-IP Complexes and 53BP2-IP1, 53BP2-IP2,and 53BP2-IP3

The invention provides for treatment or prevention of various diseasesand disorders-by administration of a therapeutic compound (termed herein“Therapeutic”). Such “Therapeutics” include but are not limited to:53BP2:53BP2-IP complexes (e.g. 53BP2 complexed with β-tubulin, p62,hnRNP G, 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3)., 53BP2 and the individual53BP2-IPs (e.g., β-tubulin, p62, hnRNP G, 53BP2-IP1, 53BP2-IP2, and53BP2-IP3) proteins and analogs and derivatives (including fragments) ofthe foregoing (e.g., as described hereinabove); antibodies thereto (asdescribed hereinabove); nucleic acids encoding the 53BP2 and/or the53BP2-IP and 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 and analogs orderivatives, thereof (e.g., as described hereinabove); 53BP2, 53BP2-IP,53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 antisense nucleic acids, and53BP2:53BP2-IP complex and 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3modulators (i.e., inhibitors, agonists and antagonists).

53BP2 and several of its binding partners, as identified herein, (e.g.β-tubulin, p62, and hnRNP G) are implicated significantly in disordersof cell cycle progression, cell differentiation, and transcriptionalcontrol, including cancer and tumorigenesis and tumor progression.Disorders of neurodegeneration resulting from altered cellularapoptosis, mRNA destabilization, and ubiquitin-mediated proteolysis, canlikewise involve these same proteins. HnRNP G is specifically implicatedin autoimmune disorders. A wide range of cell diseases affected byintracellular signal transduction, including c-Src signalling, andtranslational regulation are treated or prevented by administration of aTherapeutic that modulates (i.e. inhibits, antagonizes or promotes)53BP2:53BP2-IP complex activity, or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3activity.

Diseases and disorders associated with aberrant levels of 53BP2:53BP2-IPcomplex levels or activity or aberrant levels of 53BP2 and/or a53BP2-IP, 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 may be treated or preventedby administration of a Therapeutic that modulates 53BP2:53BP2-IP complexformation or activity or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 activity. Ina specific embodiment, the activity or levels of 53BP2 is modulated byadministration of a 53BP2-IP. In another specific embodiment, theactivity or levels of a 53BP2-IP is modulated by administration of53BP2.

Diseases and disorders characterized by increased (relative to a subjectnot suffering from the disease or disorder) 53BP2:53BP2-IP levels oractivity or increased 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 levels oractivity can be treated with Therapeutics that antagonize (i.e., reducesor inhibits) 53BP2:53BP2-IP complex formation or activity or 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 levels or activity. Therapeutics that can beused include but are not limited to 53BP2 or a 53BP2-IP or analogs,derivatives or fragments thereof, anti-53BP2:53BP2-IP complex antibodies(e.g. antibodies specific for 53BP2:β-tubulin, 53BP2:p62, 53BP2:hnRNP G,53BP2:53BP2-IP1, 53BP2:53BP2-IP2, or 53BP2-53BP2-IP3 complexes) andanti-53BP2-IP1, anti-53BP2-IP2, and anti-53BP2-IP3 antibodies (fragmentsand derivatives thereof containing the binding region thereof), nucleicacids encoding 53BP2 or 53BP2-IP, concurrent administration of 53BP2 anda 53BP2-IP antisense nucleic acid or a 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 anti-sense nucleic acid, and 53BP2 and/or 53BP2-IP, or53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 nucleic acids that are dysfunctional(e.g., due to a heterologous (non-53BP2 and/or non-53BP2-IP, ornon-53BP2-IP1, non-53BP2-IP2, or non-53BP2-IP3) insertion within the53BP2, 53BP2-IP, 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 coding sequences)that are used to “knockout” endogenous 53BP2 and/or 53BP2-IP, or53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 function by homologous recombination(see, e.g., Capecchi, 1989, Science 244: 1288-1292). In a specificembodiment of the invention, a nucleic acid containing a portion of a53BP2 and/or a 53BP2-IP or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 gene inwhich the 53BP2, 53BP2-IP, 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 sequencesflank (are both 5′ and 3′ to) a different gene sequence, is used, as a53BP2 and/or 53BP2-IP or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 antagonist,to promote 53BP2 and/or 53BP2-IP or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3inactivation by homologous recombination (see also Koller and Smithies,1989, Proc. Natl. Acad. Sci. USA 86: 8932-8935; Zijlstra et al., 1989,Nature 342: 435-438). Additionally, mutants or derivatives of a first53BP2-IP protein that have greater affinity for 53BP2 than the wild typefirst 53BP2-IP may be administered to compete with a second 53BP2-IPprotein for 53BP2 binding, thereby reducing the levels of 53BP2complexes with the second 53BP2-IP. Other Therapeutics that inhibit53BP2:53BP2-IP complex or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 functioncan be identified by use of known convenient in vitro assays, e.g.,based on their ability to inhibit 53BP2-53BP2-IP binding or as describedin Section 5.6 infra.

In specific embodiments, Therapeutics that antagonize 53BP2:53BP2-IPcomplex formation or activity or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3activity are administered therapeutically (including prophylactically):(1) in diseases or disorders involving an increased (relative to normalor desired) level of 53BP2:53BP2-IP complex or 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 proteins, for example, in patients where 53BP2:53BP2-IPcomplexes or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 is overactive oroverexpressed; or (2) in diseases or disorders wherein in vitro (or invivo) assays (see infra) indicate the utility-of 53BP2:53BP2-IP complexor 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 antagonist administration.Increased levels of 53BP2:53BP2-IP complexes or 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 protein can be readily detected, e.g., by quantifying proteinand/or RNA (or cDNA generated from RNA), by obtaining a patient tissuesample (e.g., from biopsy tissue) and assaying it in vitro for RNA orprotein levels, structure and/or activity of the expressed53BP2:53BP2-IP complex (or the 53BP2 and 53BP2-IP mRNA) or 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 protein or mRNA. Many methods standard in theart can be thus employed, including but not limited to, immunoassays todetect 53BP2:53BP2-IP complexes or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3and/or visualize 53BP2:53BP2-IP complexes or 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 protein (e.g., Western blot, immunoprecipitation followed bysodium dodecyl sulfate polyacrylamide gel electrophoresis,immunocytochemistry, etc.) and/or hybridization assays to detectconcurrent expression of 53BP2 and a 53BP2-IP, or 53BP2-IP1, 53BP2-IP2,or 53BP2-IP3 mRNA (e.g., Northern assays, dot blots, in situhybridization, etc.).

A more specific embodiment includes methods of reducing 53BP2:53BP2-IPcomplex expression (i.e., the expression of the two components of the53BP2:53BP2-IP complex and/or formation of the complex) or 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 expression, by targeting mRNAs that express theprotein moieties. RNA therapeutics currently fall within three classes,antisense species, ribozymes, or RNA aptamers (Good et al., 1997, GeneTherapy 4: 45-54). Antisense oligonucleotides have been the mode widelyused. By way of example, but not for limitation, antisenseoligonucleotide methodology to reduce 53BP2 complex formation ispresented below in subsection 5.5.7 infra. Ribozyme therapy involves theadministration, induced expression, etc. of small RNA molecules withenzymatic ability to cleave, bind, or otherwise inactivate specific RNAsto reduce or eliminate expression of particular proteins (Grassi andMarini, 1996, Annals of Medicine 28: 499-510; Gibson, 1996, Cancer andMetastasis Reviews 15: 287-299). At present, the design of “hairpin” and“hammerhead” RNA ribozymes is necessary to specifically target aparticular mRNA such as that for 53BP2. RNA aptamers are specific RNAligands for proteins, such as for Tat and Rev RNA (Good et al., 1997,Gene Therapy 4: 45-54) that can specifically inhibit their translation.Aptamers specific for 53BP2, a 53BP2-IP, 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 can be identified by many methods well known in the art, forexample but not limited to the protein-protein interaction assaydescribed in Section 5.7.1 infra.

In another embodiment, the activity or levels of 53BP2 is reduced byadministration of a 53BP2-IP, or a nucleic acid that encodes the53BP2-IP, or antibody that immunospecifically binds the 53BP2-IP, or afragment or derivative of the antibody containing the binding domainthereof. Additionally, the levels or activity of a 53BP2-IP maybereduced by administration of 53BP2 or a nucleic acid encoding 53BP2, oran antibody that immunospecifically binds 53BP2, or a fragment orderivative of the antibody containing the binding domain thereof.

In another aspect of the invention, diseases or disorders associatedwith increased levels of 53BP2 or a particular 53BP2-IP (e.g. β-tubulin,p62, hnRNP G, 53BP2-IP1, 53BP2-IP2 or 53BP2-IP3) may be treated orprevented by administration of a Therapeutic that increases53BP2:53BP2-IP complex formation if the complex formation acts to reduceor inactivate 53BP2 or the particular 53BP2-IP through the53BP2:53BP2-IP complex formation. Such diseases or disorders can betreated or prevented by administration of one member of the53BP2:53BP2-IP complex, including mutants of a member of the53BP2:53BP2-IP that has increased affinity for the other member of the53BP2:53BP2-IP complex (to cause increased complex formation),administration of antibodies or other molecules that stabilize the53BP2:53BP2-IP complex, etc.

Diseases and disorders associated with underexpression of a53BP2:53BP2-IP or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 or 53BP2 or a53BP2-IP are treated or prevented by administration of a Therapeuticthat promotes (i.e., increases or supplies) 53BP2:53BP2-IP complex or53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 function. Examples of such aTherapeutic include but are not limited to 53BP2:53BP2-IP complexes and53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 proteins and derivatives, analogsand fragments thereof that are functionally active (e.g., active to form53BP2:53BP2-IP complexes), uncomplexed 53BP2 and 53BP2-IP proteins andderivatives, analogs fragments thereon and nucleic acids encoding themembers of a 53BP2:53BP2-IP complex or encoding 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 or functionally active derivative or fragment thereof (e.g.,for use in gene therapy). In a specific embodiment, derivatives,homologs or fragments of 53BP2 and/or a 53BP2-IP that increase and/orstabilize 53BP2:53BP2-IP complex formation. Examples of other agonistscan be identified using in vitro assays or animal models, examples ofwhich are described supra.

In specific embodiments, Therapeutics that promote 53BP2:53BP2-IPcomplex or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 function are administeredtherapeutically (including prophylactically): (1) in diseases ordisorders involving an absence or decreased (relative to normal ordesired) level of 53BP2:53BP2-IP complex or 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 proteins, for example, in patients where 53BP2:53BP2-IPcomplexes (or the individual components necessary to form the complexes)or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 is lacking, genetically defective,biologically inactive or underactive, or under-expressed; or (2) indiseases or disorders wherein in vitro (or in vivo) assays (see infra)indicate the utility of 53BP2:53BP2-IP complex or 53BP2-IP1, 53BP2-IP2,53BP2-IP3 agonist administration. The absence or decreased level in53BP2:53BP2-IP complex or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 protein orfunction can be readily detected, e.g., by obtaining a patient tissuesample (e.g., from biopsy tissue) and assaying it in vitro for RNA (orcDNA generated from mRNA) or protein levels, structure and/or activityof the expressed 53BP2:53BP2-IP complex (or for the concurrentexpression of mRNA encoding the two components of the 53BP2:53BP2-IPcomplex) or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 RNA or protein. Manymethods standard in the art can be thus employed, including but notlimited to immunoassays to detect and/or visualize 53BP2:53BP2-IPcomplexes (or the individual components of 53BP2:53BP2-IP complexes) or53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 (e.g., Western blot,immunoprecipitation followed by sodium dodecyl sulfate polyacrylamidegel electrophoresis, immunocytochemistry, etc.) and/or hybridizationassays to detect expression of the mRNA encoding the individual proteincomponents of the 53BP2:53BP2-IP complexes or 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 by detecting and/or visualizing 53BP2 and a 53BP2-IPconcurrently or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 mRNA (e.g., Northernassays, dot blots, in situ hybridization, etc.), etc.

In specific embodiment, the activity or levels of 53BP2 are increased byadministration of a 53BP2-IP, or derivative or analog thereof, a nucleicacid encoding a 53BP2-IP, or an antibody that immunospecifically binds a53BP2-IP, or a fragment or derivative of the antibody contains thebinding domain thereof. In another specific embodiment, the activity orlevels of a 53BP2-IP are increased by administration of 53BP2, orderivative or analog thereof, a nucleic acid encoding 53BP2, or anantibody that immunospecifically binds 53BP2, or a fragment orderivative of the antibody contains the binding domain thereof.

Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, a human53BP2:53BP2-IP complex or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 protein, orderivative or analog thereof, nucleic acids encoding the members of thehuman 53BP2:53BP2-IP complex or human 53BP2-IP1, human 53BP2-IP2 orhuman 53BP2-IP3 or derivative or analog thereof, an antibody to a human53BP2:53BP2-IP complex or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 orderivative thereof, is therapeutically or prophylactically administeredto a human patient.

Preferably, suitable in vitro or in vivo assays, are utilized todetermine the effect of a specific Therapeutic and whether itsadministration is indicated for treatment of the affected tissue.

In various specific embodiments, in vitro assays can be carried out withrepresentative cells of cell types involved in a patient's disorder, todetermine if a Therapeutic has a desired effect upon such cell types.

Compounds for use in therapy can be tested in suitable animal modelsystems prior to testing in humans, including but not limited to rats,mice, chicken, cows, monkeys, rabbits, etc. For in vivo testing, priorto administration to humans, any animal model system known in the artmay be used. Additional descriptions and sources of Therapeutics thatcan be used according to the invention are found in Sections 5.1-5.3 and5.8 herein.

Malignancies

Components of the 53BP2:53BP-IP complexes (i.e., 53BP2, β-tubulin, p62and hnRNP G) have been implicated in regulation of cell proliferation.Accordingly, Therapeutics of the invention may be useful in treating orpreventing diseases or disorders associated with cell overproliferationor loss of control of cell proliferation, particularly cancers,malignancies and tumors. Therapeutics of the invention can be assayed byany method known in the art for efficacy in treating or preventingmalignancies and related disorders, such assays include in vitro assaysusing transformed cells or cells derived from a tumor of a patient or invivo assays using animal models of cancer or malignancies, or any of theassays described in Section 5.6 infra. Potentially effectiveTherapeutics, for example but not limited to, inhibit proliferation oftumor or transformed cells in culture or cause regression of tumors inanimal models in comparison to controls, e.g., as described in Section5.6, supra.

Accordingly, once a malignancy or cancer has been shown to be amenableto treatment by modulation (i.e., inhibit, antagonize or agonize) of53BP2:53BP2-IP complex activity or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3activity, that cancer or malignancy can be treated or prevented byadministration of a Therapeutic that modulates 53BP:53BP2-IP complexformation (including supplying 53BP2:53BP2-IP complexes and theindividual binding partners of a 53BP2:53BP2-IP complex, e.g., 53BP2,β-tubulin, p62, hnRNP G, 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3), or53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 function. Such cancer andmalignancies include but are not limited to those listed in Table 1 (fora review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed.,J. B. Lippincott Co., Philadelphia).

TABLE 1 MALIGNANCIES AND RELATED DISORDERS Leukemia acute leukemia acutelymphocytic leukemia acute myelocytic leukemia myeloblasticpromyelocytic myelomonocytic monocytic erythroleukemia chronic leukemiachronic myelocytic (granulocytic) leukemia chronic lymphocytic leukemiaPolycythemia vera Lymphoma Hodgkin's disease non-Hodgkin's diseaseMultiple myeloma Waldenström's macroglobulinemia Heavy chain diseaseSolid tumors sarcomas and carcinomas fibrosarcoma myxosarcomaliposarcoma chondrosarcoma osteogenic sarcoma chordoma angiosarcomaendotheliosarcoma lymphangiosarcoma lymphangioendotheliosarcomasynovioma mesothelioma Ewing's tumor leiomyosarcoma rhabdomyosarcomacolon carcinoma pancreatic cancer breast cancer ovarian cancer prostatecancer squamous cell carcinoma basal cell carcinoma adenocarcinoma sweatgland carcinoma sebaceous gland carcinoma papillary carcinoma papillaryadenocarcinomas cystadenocarcinoma medullary carcinoma bronchogeniccarcinoma renal cell carcinoma hepatoma bile duct carcinomachoriocarcinoma seminoma embryonal carcinoma Wilms' tumor cervicalcancer uterine cancer testicular tumor lung carcinoma small cell lungcarcinoma bladder carcinoma epithelial carcinoma glioma astrocytomamedulloblastoma craniopharyngioma ependymoma pinealoma hemangioblastomaacoustic neuroma oligodendroglioma menangioma melanoma neuroblastomaretinoblastoma

In specific embodiments, malignancy or dysproliferative changes (such asmetaplasias and dysplasias), or hyperproliferative disorders, aretreated or prevented in the bladder, breast, colon, lung, melanoma,pancreas, or uterus. In other specific embodiments, sarcoma, or leukemiais treated or prevented.

Premalignant Conditions

The Therapeutics of the invention that are effective in treating canceror malignancies (e.g. as described above) can also be administered totreat premalignant conditions and to prevent progression to a neoplasticor malignant state, including but not limited to those disorders listedin Table 1. Such prophylactic or therapeutic use is indicated inconditions known or suspected of preceding progression to neoplasia orcancer, in particular, where non-neoplastic cell growth consisting ofhyperplasia, metaplasia, or most particularly, dysplasia has occurred(for review of such abnormal growth conditions, see Robbins and Angell,1976, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp.68-79). Hyperplasia is a form of controlled cell proliferation involvingan increase in cell number in a tissue or organ, without significantalteration in structure or function. As but one example, endometrialhyperplasia often precedes endometrial cancer. Metaplasia is a form ofcontrolled cell growth in which one type of adult cell or fullydifferentiated cell substitutes for another type of adult cell.Metaplasia can occur in epithelial or connective tissue cells. Atypicalmetaplasia involves a somewhat disorderly metaplastic epithelium.Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia; it is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplastic cells often haveabnormally large, deeply stained nuclei, and exhibit pleomorphism.Dysplasia characteristically occurs where there exists chronicirritation or inflammation, and is often found in the cervix,respiratory passages, oral cavity, and gall bladder.

Alternatively or in addition to the presence of abnormal cell growthcharacterized as hyperplasia, metaplasia, or dysplasia, the presence ofone or more characteristics of a transformed phenotype, or of amalignant phenotype, displayed in vivo or displayed in vitro by a cellsample from a patient, can indicate the desirability ofprophylactic/therapeutic administration of a Therapeutic of theinvention that modulates 53BP2:53BP2-IP complex activity or 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 activity. As mentioned supra, suchcharacteristics of a transformed phenotype include morphology changes,looser substratum attachment, loss of contact inhibition, loss ofanchorage dependence, protease release, increased sugar transport,decreased serum requirement, expression of fetal antigens, disappearanceof the 250,000 dalton cell surface protein, etc. (see also id., at pp.84-90 for characteristics associated with a transformed or malignantphenotype).

In a specific embodiment, leukoplakia, a benign-appearing hyperplasticor dysplastic lesion of the epithelium, or Bowen's disease, a carcinomain situ, are pre-neoplastic lesions indicative of the desirability ofprophylactic intervention.

In another embodiment, fibrocystic disease (cystic hyperplasia, mammarydysplasia, particularly adenosis (benign epithelial hyperplasia)) isindicative of the desirability of prophylactic intervention.

In other embodiments, a patient which exhibits one or more of thefollowing predisposing factors for malignancy is treated byadministration of an effective amount of a Therapeutic: a chromosomaltranslocation associated with a malignancy (e.g., the Philadelphiachromosome for chronic myelogenous leukemia, t(14;18) for follicularlymphoma, etc.), familial polyposis or Gardner's syndrome (possibleforerunners of colon cancer), benign monoclonal gammopathy (a possibleforerunner of multiple myeloma), and a first degree kinship with personshaving a cancer or precancerous disease showing a Mendelian (genetic)inheritance pattern (e.g., familial polyposis of the colon, Gardner'ssyndrome, hereditary exostosis, polyendocrine adenomatosis, medullarythyroid carcinoma with amyloid production and pheochromocytoma,Peutz-Jeghers syndrome, neurofibromatosis of Von Recklinghausen,retinoblastoma, carotid body tumor, cutaneous melanocarcinoma,intraocular melanocarcinoma, xeroderma pigmentosum, ataxiatelangiectasia, Chediak-Higashi syndrome, albinism, Fanconi's aplasticanemia, and Bloom's syndrome; see Robbins and Angell, 1976, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 112-113) etc.)

In another specific embodiment, a Therapeutic of the invention isadministered to a human patient to prevent progression to breast, colon,lung, pancreatic, or uterine cancer, or melanoma or sarcoma.

Hyperproliferative and Dysproliferative Disorders

In another embodiment of the invention, a Therapeutic is administered totreat or prevent hyperproliferative or benign dysproliferativedisorders. Therapeutics of the invention can be assayed by any methodknown in the art for efficacy in treating or preventinghyperproliferative diseases or disorders, such assays include in vitrocell proliferation assays, in vitro or in vivo assays using animalmodels of hyperproliferative diseases or disorders, or any of the assaysdescribed in Section 5.6 infra. Potentially effective Therapeutics, forexample but not limited to, promote cell proliferation in culture orcause growth or cell proliferation in animal models in comparison tocontrols.

Accordingly, once a hyperproliferative disorder has been shown to beamenable to treatment by modulation of 53BP2:53BP2-IP complex activityor 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 activity, that hyperproliferativedisease or disorder can be treated or prevented by administration of aTherapeutic that modulates 53BP:53BP2-IP complex formation (includingsupplying 53BP2:53BP2-IP complexes and the individual binding partnersof a 53BP2:53BP2-IP complex, e.g., 53BP2, β-tubulin, p62, hnRNP G,53BP2-IP1, 53BP2-IP2 and 53BP2-IP3) or 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 function. Specific embodiments are directed to treatment orprevention of cirrhosis of the liver (a condition in which scarring hasovertaken normal liver regeneration processes), treatment of keloid(hypertrophic scar) formation (disfiguring of the skin in which thescarring process interferes with normal renewal), psoriasis (a commonskin condition characterized by excessive proliferation of the skin anddelay in proper cell fate determination), benign tumors, fibrocysticconditions, and tissue hypertrophy (e.g., prostatic hyperplasia).

Neurodegenerative Disorders

53BP2 and certain binding partners of 53BP2 (e.g. β-tubulin and p62)have been implicated in the deregulation of cellular maturation andapoptosis, which are characteristic of neurodegenerative disease.Accordingly, Therapeutics of the invention, particularly those thatmodulate (or supply) 53BP2:β-tubulin or 53BP2:p62 complexes maybeeffective in treating or preventing neurodegenerative disease.Therapeutics of the invention (particularly those that modulate thelevels or activity of 53BP2:β-tubulin or 53BP2:p62 complexes) can beassayed by any method known in the art for efficacy in treating orpreventing such neurodegenerative diseases and disorders, such assaysinclude in vitro assays for regulated cell maturation or inhibition ofapoptosis or in vivo assays using animal models of neurodegenerativediseases or disorders, or any of the assays described in Sections 5.6infra. Potentially effective Therapeutics, for example but not by way oflimitation, promote regulated cell maturation and prevent cell apoptosisin culture or reduce neurodegeneration in animal models in comparison tocontrols.

Once a neurodegenerative disease or disorder has been shown to beamenable to treatment by modulation of 53BP2:53BP2-IP complex activityor 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 activity, that neurodegenerativedisease or disorder can be treated or prevented by administration of aTherapeutic that modulates 53BP:53BP2-IP complex formation (includingsupplying 53BP2:53BP2-IP complexes, e.g., 53BP2: β-tubulin and 53BP2:p62complexes) or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 function.

Such diseases include the neurodegenerative disorders involved withaging, especially osteoarthritis and neurodegenerative disorders.Neurodegenerative disorders that can be treated or prevented include butare not limited to those listed in Table 2 (see Isslebacher et al.,1997, in: Harrison's Principals of Internal Medicine, 13^(th) Ed.,McGraw Hill, New York).

TABLE 2 NEURODEGENERATIVE DISORDERS Progressive dementia in the absenceof other neurological signs Alzheimer's Disease (or early-onset AD)Senile dementia of the Alzheimer's type (or late onset AD) Pick'sDisease Syndromes combining progressive dementia with prominentneurological abnormalities Huntington's disease Multiple system atrophy(dementia combined with ataxia, Parkinson's disease, etc.) Progressivesupranuclear palsy Diffuse Lewy body disease Corticodentatonigraldegeneration Hallervorden-Spatz disease Progressive familial myoclonicepilepsy Syndromes of gradually developing abnormalities of posture andmovement Parkinson's disease Striatonigral degeneration Progressivesupranuclear palsy Torsion dystonia Spasmodic torticollis and otherrestricted dyskinesias Familial tremor Gilles de la Tourette syndromeSyndromes of progressive ataxia Cerebellar cortical degenerationOlivopontocerebellar atrophy Friedrich's ataxia and relatedspinocerebellar degenerations Shy-Drager syndrome Subacute necrotizingencephalopathy Motor neuron disease without sensory changes Amyotrophiclateral sclerosis Infantile spinal muscular atrophy Juvenile spinalmuscular atrophy Other forms of familial spinal muscular atrophy Primarylateral sclerosis Hereditary spastic paraplegia Motor neuron diseasewith sensory changes Peroneal muscular atrophy Hypertrophic interstitialpolyneuropathy Other forms of chronic progressive neuropathy Syndromesof progressive visual loss Retinitis pigmentosa

Autoimmune Disorders

The 53BP2 binding partner hnRNP G has been implicated in autoimmunedisorders. Therapeutics of the invention, particularly those thatmodulate (or supply) 53BP2:hnRNPG complex activity may be effective intreating or preventing autoimmune diseases or disorders. Therapeutics ofthe invention (particularly Therapeutics that modulate the levels oractivity of 53BP2:hnRNP G) can be assayed by any method known in the artfor efficacy in treating or preventing such autoimmune diseases anddisorders, such assays include in vitro assays for using cell culturemodels as described in Section 5.6, infra, or in vivo assays usinganimal models of autoimmune diseases or disorders as described inSection 5.6 infra. Potentially effective Therapeutics, for example butnot by way of limitation, reduce autoimmune responses in animal modelsin comparison to controls.

Accordingly, once an autoimmune disease or disorder has been shown to beamenable to treatment by modulation of 53BP2:53BP2-IP complex activityor 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 activity, that autoimmune diseaseor disorder can be treated or prevented by administration of aTherapeutic that modulates 53BP:53BP2-IP complex formation (includingsupplying 53BP2:53BP2-IP complexes) or 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 function.

Autoimmune disorders that can be treated or prevented include but arenot limited to those listed in Table 3 (Isslebacher et al., 1997, inHarrison's Principals of Internal Medicine, 13^(th) Ed., McGraw Hill,New York).

TABLE 3 AUTOIMMUNE DISEASES Organ Specific Endocrine thyroid Hashimoto'sthyroiditis Grave's disease thyroiditis with hyperthyroidism type Iautoimmune polyglandular syndrome type II autoimmune polyglandularsyndrome insulin-dependent diabetes mellitus immune-mediated infertilityautoimmune Addison's disease Skin pemphigus vulgaris pemphigus foliaceusbullus pemphigoid dermatitis herpetiformis linear IgA dermatosisepidermolysis bullous aquisita autoimmune allopecia erythema nodosacontact dermatitis herpes gestationis cicatricial pemphigoid chronicbullous disease of childhood Hemotologic autoimmune hemolytic anemialymphomas chronic lymphocytic anemia non-Hodgkin's lymphoma Hodgkin'sdisease drug-induced alpha methyl dopa penicillin type quinidine typepost-viral infections tumors (rare) cold agglutinin diseases acutemycoplasma infection infectious mononucleosis chronic idiopathiclymphoma paroxysmal cold hemoglobinuria autoimmune thrombocytopenicpurpura idiopathic drug-induced autoimmune neutropenia Neuromuscularmyasthenia gravis acute disseminated encephalomyelitis multiplesclerosis Guillain-Barre syndrome Chronic inflammatory demyelinatingpolyradiculoneuropathy Hepatobiliary autoimmune chronic active hepatitisprimary biliary sclerosis sclerosing cholangitis Gastrointestinalgluten-sensitive enteropathy perniciuos anemia inflammatory boweldisease Non-Organ Specific Connective tissue disease system lupuserythematososis rheumatoid arthritis scleroderma mixed connective tissuedisease psoriasis polymyositis dermatomyositis Sjogren's syndromeankylosing spondylitis reactive arthritis undifferentiatedspondylarthropathy Behcet's syndrome Vasculitis syndromes systemicnecrotizing vasculitides classic polyarteritis nodosa Churg-Straussdisease Polyangiitis overlap syndrome hypersensitivity vasculitisWegener's granulomatosis temporal arteritis Takayasu's arteritisKawasaki's disease isolated vasculitis of the central nervous systemthromboangiitis obliterans Sarcoidosis Graft-vs-host disease

Gene Therapy

In a specific embodiment, nucleic acids comprising a sequence encoding a53BP2 and a 53BP2-IP, or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3, orfunctional derivatives thereof, are administered to modulate53BP2:53BP2-IP complex or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 function,by way of gene therapy. In more specific embodiments, a nucleic acid ornucleic acids encoding both 53BP2 and a 53BP2-IP (e.g., β-tubulin, p62,hnRNP G, 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3) or functional derivativesthereof, are administered by way of gene therapy. Gene therapy refers totherapy performed by the administration of a nucleic acid to a subject.In this embodiment of the invention, the nucleic acid produces itsencoded protein(s) that mediates a therapeutic effect by modulating53BP2:53BP2-IP complex or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 function.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., 1993, Clinical Pharmacy 12: 488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32: 573-596;Mulligan, 1993, Science 260: 926-932; and Morgan and Anderson, 1993,Ann. Rev. Biochem. 62: 191-217; May, 1993, TIBTECH 11(5): 155-215).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds.), 1993, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY; and Kriegler,1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press,NY.

In a preferred aspect, the Therapeutic comprises a 53BP2 and a 53BP2-IPnucleic acid or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 nucleic acid that ispart of an expression vector that expresses the proteins 53BP2 and a53BP2-IP or expresses 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 or fragments orchimeric proteins thereof in a suitable host. In particular, such anucleic acid has a promoter operably linked to the 53BP2 and the53BP2-IP coding region(s) (or, less preferably two separate promoterslinked to the 53BP2 and the 53BP2-IP coding regions separately) orlinked to the 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 coding region, saidpromoter being inducible or constitutive, and, optionally,tissue-specific. In another particular embodiment, a nucleic acidmolecule is used in which the 53BP2 and 53BP2-IP coding sequences or53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 coding sequences, and any otherdesired sequences, are flanked by regions that promote homologousrecombination at a desired site in the genome, thus providing forintra-chromosomal expression of the 53BP2 and the 53BP2-IP nucleic acidsor 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 nucleic acids (Koller andSmithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra etal., 1989, Nature 342:435-438).

Delivery of the nucleic acid into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vector, or indirect, in which case, cells arefirst transformed with the nucleic acid in vitro, then transplanted intothe patient. These two approaches are known, respectively, as in vivo orex vivo gene therapy.

In a specific embodiment, the nucleic acid is directly administered invivo, where it is expressed to produce the encoded product. This can beaccomplished by any of numerous methods known in the art, e.g., byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular, e.g., byinfection using a defective or attenuated retroviral or other viralvector (see U.S. Pat. No. 4,980,286), or by direct injection of nakedDNA, or by use of microparticle bombardment (e.g., a gene gun;Biolistic, Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, encapsulation in liposomes, microparticles, ormicrocapsules, or by administering it in linkage to a peptide which isknown to enter the nucleus, by administering it in linkage to a ligandsubject to receptor-mediated endocytosis (see e.g., Wu and Wu, 1987, J.Biol. Chem. 262:4429-4432) (which can be used to target cell typesspecifically expressing the receptors), etc. In another embodiment, anucleic acid-ligand complex can be formed in which the ligand comprisesa fusogenic viral peptide to disrupt endosomes, allowing the nucleicacid to avoid lysosomal degradation. In yet another embodiment, thenucleic acid can be targeted in vivo for cell specific uptake andexpression, by targeting a specific receptor (see, e.g., PCTPublications WO 92/06180 dated Apr. 16, 1992 (Wu et al.); WO 92/22635dated Dec. 23, 1992 (Wilson et al.); WO92/20316 dated Nov. 26, 1992(Findeis et al.); WO93/14188 dated Jul. 22, 1993 (Clarke et al.), WO93/20221 dated Oct. 14, 1993 (Young)). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, 1989,Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature342:435-438).

In a specific embodiment, a viral vector that contains the 53BP2 and/orthe 53BP2-IP nucleic acids or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 nucleicacid is used. For example, a retroviral vector can be used (see Milleret al., 1993, Meth. Enzymol. 217:581-599). These retroviral vectors havebeen modified to delete retroviral sequences that are not necessary forpackaging of the viral genome and integration into host cell DNA. The53BP2 and/or 53BP2-IP (preferably both 53BP2 and 53BP2-IP) nucleic acidsor 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 nucleic acids, to be used in genetherapy is/are cloned into the vector, which facilitates delivery of thegene into a patient. More detail about retroviral vectors can be foundin Boesen et al., 1994, Biotherapy 6: 291-302, which describes the useof a retroviral vector to deliver the mdr1 gene to hematopoetic stemcells in order to make the stem cells more resistant to chemotherapy.Other references illustrating the use of retroviral vectors in genetherapy are: Clowes et al., 1994, J. Clin. Invest. 93: 644-651; Kiem etal., 1994, Blood 83: 1467-1473; Salmons and Gunzberg, 1993, Human GeneTherapy 4: 129-141; and Grossman and Wilson, 1993, Curr. Opin. inGenetics and Devel. 3: 110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, 1993,Current Opinion in Genetics and Development 3: 499-503 present a reviewof adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy5: 3-10 demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252: 431-434; Rosenfeld et al., 1992, Cell 68: 143-155; andMastrangeli et al., 1993, J. Clin. Invest. 91: 225-234.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204: 289-300.

Another approach to gene therapy involves transferring a gene into cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth.Enzymol. 217: 599-618; Cohen et al., 1993, Meth. Enzymol. 217: 618-644;Cline, 1985, Pharmac. Ther. 29: 69-92) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. In a preferred embodiment, epithelial cellsare injected, e.g., subcutaneously. In another embodiment, recombinantskin cells may be applied as a skin graft onto the patient. Recombinantblood cells (e.g., hematopoetic stem or progenitor cells) are preferablyadministered intravenously. The amount of cells envisioned for usedepends on the desired effect, patient state, etc., and can bedetermined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoetic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy, a53BP2 and/or a 53BP2-IP (preferably both a 53BP2 and a 53BP2-IP) nucleicacid or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 nucleic acid is/areintroduced into the cells such that the gene.or genes are expressible bythe cells or their progeny, and the recombinant cells are thenadministered in vivo for therapeutic effect. In a specific embodiment,stem or progenitor cells are used. Any stem and/or progenitor cellswhich can be isolated and maintained in vitro can potentially be used inaccordance with this embodiment of the present invention. Such stemcells include but are not limited to hematopoetic stem cells (HSC), stemcells of epithelial tissues such as the skin and the lining of the gut,embryonic heart muscle cells, liver stem cells (PCT Publication WO94/08598, dated Apr. 28, 1994), and neural stem cells (Stemple andAnderson, 1992, Cell 71: 973-985).

Epithelial stem cells (ESCs) or keratinocytes can be obtained fromtissues such as the skin and the lining of the gut by known procedures(Rheinwald, 1980, Meth. Cell Bio. 21A: 229). In stratified epithelialtissue such as the skin, renewal occurs by mitosis of stem cells withinthe germinal layer, the layer closest to the basal lamina. Stem cellswithin the lining of the gut provide for a rapid renewal rate of thistissue. ESCs or keratinocytes obtained from the skin or lining of thegut of a patient or donor can be grown in tissue culture (Rheinwald,1980, Meth. Cell Bio. 21A: 229; Pittelkow and Scott, 1986, Mayo ClinicProc. 61: 771). If the ESCs are provided by a donor, a method forsuppression of host versus graft reactivity (e.g., irradiation, drug orantibody administration to promote moderate immunosuppression) can alsobe used.

With respect to hematopoetic stem cells (HSC), any technique whichprovides for the isolation, propagation, and maintenance in vitro of HSCcan be used in this embodiment of the invention. Techniques by whichthis may be accomplished include (a) the isolation and establishment ofHSC cultures from bone marrow cells isolated from the future host, or adonor, or (b) the use of previously established long-term HSC cultures,which may be allergenic or xenogeneic. Non-autologous HSC are usedpreferably in conjunction with a method of suppressing transplantationimmune reactions of the future host/patient. In a particular embodimentof the present invention, human bone marrow cells can be obtained fromthe posterior iliac crest by needle aspiration (see, e.g., Kodo et al.,1984, J. Clin. Invest. 73: 1377-1384). In a preferred embodiment of thepresent invention, the HSCs can be made highly enriched or insubstantially pure form. This enrichment can be accomplished before,during, or after long-term culturing, and can be done by any techniquesknown in the art. Long-term cultures of bone marrow cells can beestablished and maintained by using, for example, modified Dexter cellculture techniques (Dexter et al., 1977, J. Cell Physiol. 91: 335) orWitlock-Witte culture techniques (Witlock and Witte, 1982, Proc. Natl.Acad. Sci. USA 79: 3608-3612).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Additional methods can be adapted for use to deliver a nucleic acidencoding the 53BP2 and/or 53BP2-IP proteins or 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 proteins, or functional derivatives thereof, e.g., asdescribed in Sections 5.1 and 5.2 supra.

Use of Antisense Oligonucleotides for Suppression of 53BP2:53BP2-IPComplexes and 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3

In a specific embodiment, 53BP2:53BP2-IP complex function or 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 protein function is inhibited by use ofantisense nucleic acids for 53BP2 and/or a 53BP2-IP (e.g., β-tubulin,p62, hnRNP G, 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3) (preferably both53BP2 and the 53BP2-IP) or antisense nucleic acids for 53BP2-IP1,53BP2-IP2, or 53BP2-IP3. The present invention provides the therapeuticor prophylactic use of nucleic acids of at least six nucleotides thatare antisense to a gene or cDNA encoding 53BP2 and/or a 53BP2-IP orencoding 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3, or portions thereof. A53BP2, 53BP2-IP, 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 “antisense” nucleicacid as used herein refers to a nucleic acid capable of hybridizing to aportion of a 53BP2, 53BP2-IP, 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 RNA(preferably mRNA) by virtue of some sequence complementarity. Theantisense nucleic acid may be complementary to a coding and/or noncodingregion of a 53BP2, 53BP2-IP, 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 mRNA.Such antisense nucleic acids have utility as Therapeutics that inhibit53BP2:53BP2-IP complex formation or activity or 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 function or activity, and can be used in the treatment orprevention of disorders as described supra.

The antisense nucleic acids of the invention can be oligonucleotidesthat are double-stranded or single-stranded, RNA or DNA or amodification or derivative thereof, which can be directly administeredto a cell, or which can be produced intracellularly by transcription ofexogenous, introduced sequences.

In another embodiment, the invention is directed to methods forinhibiting the expression of 53BP2 and a 53BP2-IP nucleic acid sequenceor 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 nucleic acid sequence in aprokaryotic or eukaryotic cell comprising providing the cell with aneffective amount of a composition comprising an antisense nucleic acidof 53BP2 and 53BP2-IP, or an antisense nucleic acid of 53BP2-IP1,53BP2-IP2, or 53BP2-IP3, or derivatives thereof, of the invention.

The 53BP2, 53BP2-IP, 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 antisensenucleic acids are of at least six nucleotides and are preferablyoligonucleotides (ranging from 6 to about 200 oligonucleotides). Inspecific aspects, the oligonucleotide is at least 10 nucleotides, atleast 15 nucleotides, at least 100 nucleotides, or at least 200nucleotides. The oligonucleotides can be DNA or RNA or chimeric mixturesor derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone. The oligonucleotide may includeother appending groups such as peptides, or agents facilitatingtransport across the cell membrane (see, e.g., Letsinger et al., 1989,Proc. Natl. Acad. Sci. U.S.A. 86: 6553-6556; Lemaitre et al., 1987,Proc. Natl. Acad. Sci. 84: 648-652; PCT Publication No. WO 88/09810,published Dec. 15, 1988) or blood-brain barrier (see, e.g., PCTPublication No. WO 89/10134, published Apr. 25, 1988),hybridization-triggered cleavage agents (see, e.g., Krol et al., 1988,BioTechniques 6: 958-976) or intercalating agents (see, e.g., Zon, 1988,Pharm. Res. 5: 539-549).

In a preferred aspect of the invention, a 53BP2, 53BP2-IP, 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 antisense oligonucleotide is provided,preferably as single-stranded DNA. The oligonucleotide may be modifiedat any position on its structure with constituents generally known inthe art.

The 53BP2, 53BP2-IP, 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 antisenseoligonucleotides may comprise at least one modified base moiety which isselected from the group including but not limited to 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine.

In another embodiment, the oligonucleotide comprises at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the oligonucleotide comprises at least onemodified phosphate backbone selected from the group consisting of aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the oligonucleotide is an 2-α-anomericoligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15: 6625-6641).

The oligonucleotide may be conjugated to another molecule, e.g., apeptide, hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

Oligonucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16: 3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85: 7448-7451), etc.

In a specific embodiment, the 53BP2, 53BP2-IP, 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 antisense oligonucleotides comprise catalytic RNAs, orribozymes (see, e.g., PCT International Publication WO 90/11364,published Oct. 4, 1990; Sarver et al., 1990, Science 247: 1222-1225). Inanother embodiment, the oligonucleotide is a 2′-0-methylribonucleotide(Inoue et al., 1987, Nucl. Acids Res. 15: 6131-6148), or a chimericRNA-DNA analog (Inoue et al., 1987, FEBS Lett. 215: 327-330).

In an alternative embodiment, the 53BP2, 53BP2-IP, 53BP2-IP1, 53BP2-IP2,or 53BP2-IP3 antisense nucleic acids of the invention are producedintracellularly by transcription from an exogenous sequence. Forexample, a vector can be introduced in vivo such that it is taken up bya cell, within which cell the vector or a portion thereof istranscribed, producing an antisense nucleic acid (RNA) of the invention.Such a vector would contain a sequence encoding 53BP2, 53BP2-IP(preferably, a 53BP2 and a 53BP2-IP anti-sense nucleic acid), 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 antisense nucleic acids. Such a vector canremain episomal or become chromosomally integrated, as long as it can betranscribed to produce the desired antisense RNA. Such vectors can beconstructed by recombinant DNA technology methods standard in the art.Vectors can be plasmid, viral, or others known in the art, used forreplication and expression in mammalian cells. Expression of thesequences encoding the 53BP2, 53BP2-IP, 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 antisense RNAs can be by any promoter known in the art to actin mammalian, preferably human, cells. Such promoters can be inducibleor constitutive. Such promoters include but are not limited to: the SV40early promoter region (Bernoist and Chambon, 1981, Nature 290: 304-310),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., 1980, Cell 22: 787-797), the herpes thymidinekinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al., 1982, Nature 296: 39-42), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a 53BP2,53BP2-IP, 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 gene, preferably a human53BP2, 53BP2-IP, 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 gene. However,absolute complementarity, although preferred, is not required. Asequence “complementary to at least a portion of an RNA,” as referred toherein, means a sequence having sufficient complementarity to be able tohybridize with the RNA, forming a stable duplex; in the case ofdouble-stranded 53BP2, 53BP2-IP, 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3antisense nucleic acids, a single strand of the duplex DNA may thus betested, or triplex formation may be assayed. The ability to hybridizewill depend on both the degree of complementarity and the length of theantisense nucleic acid. Generally, the longer the hybridizing nucleicacid, the more base mismatches with a 53BP2, 53BP2-IP, 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 RNA it may contain and still form a stableduplex (or triplex, as the case may be). One skilled in the art canascertain a tolerable degree of mismatch by use of standard proceduresto determine the melting point of the hybridized complex.

The 53BP2 and 53BP2-IP antisense nucleic acid or 53BP2-IP1, 53BP2-IP2,or 53BP2-IP3 antisense nucleic acids can be used to treat (or prevent)disorders of a cell type that expresses, or preferably overexpresses,the 53BP2:53BP2-IP complex or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3protein. In a preferred embodiment, a single-stranded DNA antisense53BP2 and 53BP2-IP antisense oligonucleotide or single-stranded DNAantisense 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 oligonucleotide is used.

Cell types that express or overexpress 53BP2 and 53BP2-IP RNA or53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 RNA can be identified by variousmethods known in the art. Such methods include, but are not limited to,hybridization with 53BP2- and 53BP2-IP-specific nucleic acids or53BP2-IP1-, 53B2-IP2- or 53BP2-IP3-specific nucleic acids (e.g. bynorthern hybridization, dot blot hybridization, in situ hybridization),or by observing the ability of RNA from the cell type to be translatedin vitro into 53BP2 and the 53BP2-IP or into 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 by immunohistochemistry. In a preferred aspect, primary tissuefrom a patient can be assayed for 53BP2 and 53BP2-IP expression or for53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 expression prior to treatment, e.g.,by immunocytochemistry or in situ hybridization.

Pharmaceutical compositions of the invention (see Section 5.8 infra),comprising an effective amount of a 53BP2 and a 53BP2-IP antisensenucleic acid or a 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 antisense nucleicacid in a pharmaceutically acceptable carrier, can be administered to apatient having a disease or disorder which is of a type that expressesor overexpresses 53BP2:53BP2-IP complexes or 53BP2-IP1, 53B2-IP2, or53BP2-IP3 RNA or protein.

The amount of 53BP2 and 53BP2-IP antisense nucleic acids or 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 antisense nucleic acid that will be effective inthe treatment of a particular disorder or condition will depend on thenature of the disorder or condition, and can be determined by standardclinical techniques. Where possible, it is desirable to determine theantisense cytotoxicity in vitro, and then in useful animal model systemsprior to testing and use in humans.

In a specific embodiment, pharmaceutical compositions comprising 53BP2and 53BP2-IP antisense nucleic acids or 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 antisense nucleic acids are administered via liposomes,microparticles, or microcapsules. In various embodiments of theinvention, it may be useful to use such compositions to achievesustained release of the 53BP2 and 53BP2-IP antisense nucleic acids or53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 antisense nucleic acids. In aspecific embodiment, it may be desirable to utilize liposomes targetedvia antibodies to specific identifiable central nervous system celltypes (Leonetti et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2448-2451; Renneisen et al., 1990, J. Biol. Chem. 265: 16337-16342).

Assays of 53BP2:53BP2-IP Complexes, 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3and Derivatives and Analogs

The functional activity of 53BP2:53BP2-IP complexes and 53BP2-IP1,53BP2-IP2 and 53BP2-IP3 proteins, and derivatives, fragments and analogsthereof can be assayed by various methods. Potential modulators (e.g.,inhibitors, agonists and antagonists) of 53BP2:53BP2 complex activity or53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 activity, e.g., anti-53BP2:53BP2-IP,anti-53BP2-IP1, anti-53BP2-IP2, and anti-53BP2-IP3 antibodies and 53BP2,53BP2-IP, 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 antisense nucleic acidscan be assayed for the ability to modulate 53BP2:53BP2-IP complexformation and/or activity or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3activity.

For example, in one embodiment, where one is assaying for the ability tobind or compete with wild-type 53BP2:53BP2-IP complexes or 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 for binding to an anti-53BP2:53BP2-IP antibodyor anti-53BP2-IP1, anti-53BP2-IP2, or anti-53BP2-IP3 antibodies, variousimmunoassays known in the art can be used, including but not limited tocompetitive and non-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitinreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labelled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention.

The expression of the 53BP2, 53BP2-IP, 53BP2-IP1, 53BP2-IP2, and53BP2-IP3 genes (both endogenous genes and those expressed from clonedDNA containing these genes) can be detected using techniques known inthe art, including but not limited to Southern hybridization (Southern,1975, J. Mol. Biol. 98: 503-517), northern hybridization (e.g. Freemanet al., 1983, Proc. Natl. Acad. Sci. USA 80: 4094-4098), restrictionendonuclease mapping (Sambrook et al., 1982, Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, New York), and DNA sequenceanalysis. Polymerase chain reaction amplification (PCR; U.S. Pat. Nos.4,683,202, 4,683,195, and 4,889,818; Gyllenstein et al., 1988, Proc.Natl. Acad. Sci. USA 85: 7652-7657; Ochman et al., 1988, Genetics 120:621-623; Loh et al., 1989, Science 243: 217-220) followed by Southernhybridization or RNase protection (Current Protocols in MolecularBiology, John Wiley and Sons, New York, 1997) with probes specific for53BP2, the 53BP2-IP, or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 genes invarious cell types. Methods of amplification other than PCR commonlyknown in the art can be employed. In one embodiment, Southernhybridization can be used to detect genetic linkage of 53BP2, 53BP2-IP,53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 gene mutations to physiological orpathological states. Various cell types, at various stages ofdevelopment, can be characterized for their expression of 53BP2 and a53BP2-IP (particularly expression of 53BP2 and 53BP2-IP at the same timeand in the same cells), or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3expression. The stringency of the hybridization conditions for northernor Southern blot analysis can be manipulated to ensure detection ofnucleic acids with the desired degree of relatedness to the specificprobes used. Modifications to these methods and other methods commonlyknown in the art can be used.

Derivatives (e.g., fragments) and analogs of 53BP2-IPs, including53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 (and fragments and other derivativesand analogs of 53BP2-IPs) can be assayed for binding to 53BP2 by anymethod-known in the art, for example the modified yeast two hybrid assaysystem described in Section 5.7.1 infra, immunoprecipitation with anantibody that binds to 53BP2 in a complex followed by analysis by sizefractionation of the immunoprecipitated proteins (e.g. by denaturing ornondenaturing polyacrylamide gel electrophoresis), Western analysis,non-denaturing gel electrophoresis, etc.

One embodiment of the invention provides a method for screening aderivative or analog of 53BP2 for biological activity comprisingcontacting said derivative or analog of 53BP2 with a protein selectedfrom the group consisting of β-tubulin, p62, hnRNP G, 53BP2-IP1,53BP2-IP2, and 53BP2-IP3; and detecting the formation of a complexbetween said derivative or analog or 53BP2 and said protein; whereindetecting formation of said complex indicates that said derivative oranalog of 53BP2 has biological (e.g., binding) activity. Additionally,another embodiment of the invention relates to a method for screening aderivative or analog of a protein selected from the group consisting ofβ-tubulin, p62, hnRNP G, 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 forbiological activity comprising contacting said derivative or analog ofsaid protein with 53BP2; and detecting the formation of a complexbetween said derivative or analog of said protein and 53BP2; whereindetecting the formation of said complex indicates that said derivativeor analog of said protein has biological activity.

The invention also provides methods of modulating the activity of aprotein that can participate in a 53BP2:53BP2-IP complex (e.g. 53BP2,β-tubulin, p62, hnRNP G. 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3) byadministration of a binding partner of that protein or derivative oranalog thereof. 53BP2, and derivatives and analogs thereof, can beassayed for the ability to modulate the activity or levels of a 53BP2-IPby contacting a cell or administering an animal expressing a 53BP2-IPgene with a 53BP2 protein, or a nucleic acid encoding a 53BP2 protein oran antibody that immunospecifically binds the 53BP2 protein or afragment or derivative of said antibody containing the binding domainthereof and measuring a change in 53BP2-IP levels or activity, wherein achange in 53BP2-IP levels or activity indicates that 53BP2 can modulate53BP2-IP levels or activity. Alternatively, a 53BP2-IP can be assayedfor the ability to modulate the activity or levels of a 53BP2 protein bycontacting a cell or administering an animal expressing a gene encodingsaid protein with 53BP2, or a nucleic acid encoding 53BP2, or anantibody that immunospecifically binds 53BP2, or a fragment orderivative of said antibody containing the binding domain thereof,wherein a change in 53BP2 levels or activity indicates that the 53BP2-IPcan modulate 53BP2 levels or activity.

53BP2, and several of the identified binding partners of 53BP2, e.g.β-tubulin, p62 protein and hnRNP G have roles in the control of cellproliferation and, therefore, cell-transformation and tumorigenesis.Accordingly, methods of the invention are provided for screening53BP2:53BP2-IP complexes and 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3proteins, and fragments, derivatives and analogs of the foregoing, foractivity in altering cell proliferation, cell transformation and/ortumorigenesis in vitro and in vivo.

The 53BP2:53BP2-IP complexes, 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3proteins, and derivatives, fragments, and analogs thereof, can beassayed for activity to alter (i.e., increase or decrease) cellproliferation in cultured cells in vitro using methods which arewell-known in the art for measuring cell proliferation. Specificexamples of cell culture models include, but are not limited to: forlung cancer primary rat lung tumor cells (Swafford et al., 1997, Mol.Cell. Biol., 17:1366-1374) and large-cell undifferentiated cancer celllines (Mabry et al., 1991, Cancer Cells, 3:53-58); colorectal cell linesfor colon cancer (Park and Gazdar, 1996, J. Cell Biochem. Suppl.24:131-141); multiple established cell lines for breast cancer (Hamblyet al., 1997, Breast Cancer Res. Treat. 43:247-258; Gierthy et al.,1997, Chemosphere 34:1495-1505; Prasad and Chiurch, 1997, Biochem.Biophys. Res. Commun. 232:14-19); a number of well-characterized cellmodels for prostate cancer (Webber et al., 1996, Prostate, Part 1,29:386-394; Parts 2 and 3, 30:58-64 and 136-142; Boulikas, 1997,Anticancer Res. 17:1471-1505); continuous human bladder cancer celllines for genitourinary cancers (Ribeiro et al., 1997, Int. J. Radiat.Biol. 72:11-20), organ cultures of transitional cell carcinomas (Boothet al., 1997, Lab Invest. 76:843-857), and rat progression models (Vetet al., 1997, Biochim. Biophys Acta 1360:39-44); established cell linesfor leukemias and lymphomas (Drexler, 1994, Leuk. Res. 18:919-927;Tohyama, 1997, Int. J. Hematol. 65:309-317).

For example, but not by way of limitation, cell proliferation can beassayed by measuring ³H-thymidine incorporation, by direct cell count,by detecting changes in transcriptional activity of known genes such asproto-oncogenes (e.g., fos, myc) or cell cycle markers, etc.Accordingly, one embodiment of the invention provides a method ofscreening 53BP2:53BP2-IP complexes, 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3proteins, and fragments, derivatives, and analogs thereof, for activityin altering (i.e., increasing or decreasing) proliferation of cells invitro comprising contacting the cells with a 53BP2:53BP2-IP complex or53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 protein, or derivative, analog, orfragment thereof, measuring the proliferation of cells that have been socontacted, and comparing the proliferation of the cells so contactedwith a complex or protein of the invention with the proliferation ofcells not so contacted with the complex or protein of the invention,wherein in a change in the level of proliferation in said contactedcells indicates that the complex or protein of the invention hasactivity to alter cell proliferation.

The 53BP2:53BP2-IP complexes and 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3proteins, and derivatives, fragments and analogs, thereof, can also bescreened for activity in inducing or inhibiting cell transformation (orprogression to malignant phenotype) in vitro. The complexes and proteinsof the invention can be screened by contacting either cells with anormal phenotype (for assaying for cell transformation) or a transformedcell phenotype (for assaying for inhibition of cell transformation) withthe complex or protein of the invention and examining the cells foracquisition or loss of characteristics associated with a transformedphenotype (a set of in vitro characteristics associated with atumorigenic ability in vivo), for example, but not limited to, colonyformation in soft agar, a more rounded cell morphology, loosersubstratum attachment, loss of contact inhibition, loss of anchoragedependence, release of proteases such as plasminogen activator,increased sugar transport, decreased serum requirement, expression offetal antigens, disappearance of the 250,000 dalton surface protein,etc. (see Luria et al., 1978, General Virology, 3d Ed., John Wiley &Sons, New York, pp. 436-446).

The 53BP2:53BP2-IP complexes and 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3proteins, and derivatives, fragments, and analogs thereof can also bescreened for activity to promote or inhibit tumor formation in vivo innon-human test animal. A vast number of animal models ofhyperproliferative disorders, including tumorigenesis and metastaticspread, are known in the art (see Table 317-1, Chapter 317, “Principalsof Neoplasia,” in Harrison's Principals of Internal Medicine, 13thEdition, Isselbacher et al., eds., McGraw-Hill, New York, p.1814;Lovejoy et al., 1997, J. Pathol. 181:130-135). Specific examplesinclude: transplantation of tumor modules into rats for lung cancer(Wang et al., 1997, Ann. Thorac. Surg. 64:216-219) or establishment oflung cancer metastases in SCID mice depleted of NK cells (Yono and Sone,1997, Gan To Kagaku Ryoho, 24:489-494); colon cancer transplantation ofhuman colon cancer cells into nude mice (Gutman and Fidler, 1995, WorldJ. Surg., 19:226-234), the cotton top tamarin model of human ulcerativecolitis (Warren, 1996, Aliment. Pharmacol. Ther., 10 Suppl2:45-47) andmouse models with mutations of the adenomatous polyposis coli tumorsuppressor (Polakis, 1997, Biochim. Biophys. Acta 1332:F127-F147); forbreast cancer, transgenic models of breast cancer (Dankort and Muller,1996, Cancer Treat. Res. 83:71-88; Amundadittir et al., 1996, BreastCancer Res. Treat. 39:119-135) and chemical induction of tumors in rats(Russo and Russo, 1996, Breast Cancer Res. Treat. 39:7-20); for prostatecancer, chemically-induced and transgenic rodent models, and humanxenograft models (Royai et al., 1996, Semin. Oncol. 23:35-4-0); forgenitourinary cancers, induced bladder neoplasm in rats and mice (Oyasu,1995, Food Chem. Toxicol 33:747-755) and xenografts of humantransitional cell carcinomas into nude rats (Jarrett et al., 1995, J.Endourol. 9:1-7); for hematopoietic cancers, transplanted allogeneicmarrow in animals (Appelbaum, 1997, Leukemia 11 Suppl 4:S15-S17).Further, general animal models applicable to many types of cancer havebeen described, including but not restricted to the p53-deficient mousemodel (Donehower, 1996, Semin. Cancer Biol. 7:269-278), the Min mouse(Shoemaker et al., 1997, Biochem. Biophys. Acta, 1332:F25-F48), andimmune response to tumors in the rat (Frey, 1997, Methods, 12:173-188).

For example, the complexes and proteins of the invention can beadministered to a non-human test animal (preferably a test animalpredisposed to develop a type of tumor) and the non-human test animalssubsequently examined for an increased incidence of tumor formation incomparison with controls not administered the complex or protein of theinvention. Alternatively, the complexes and proteins of the inventioncan be administered to non-human test animals having tumors (e.g.,animals in which tumors have been induced by introduction of malignant,neoplastic, or transformed cells or by administration of a carcinogen)and subsequently examining the tumors in the test animals for tumorregression in comparison to controls.

The 53BP2:53BP2-IP complexes and 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3proteins, and derivatives, analogs, and fragments thereof, can also bescreened for activity in modulating the activity of 53BP2 and the 53BP2binding partners (i.e., the 53BP2-IPs, particularly β-tubulin, p62,hnRNP G, 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3) involved in particular53BP2:53BP2-IP complexes. For example, 53BP2 has been shown to bind aspecific domain of the p53 protein and, by virtue of 53BP2-binding,enhance the tumor suppressor activity of p53. Accordingly, the complexesand proteins of the invention can be screened for the ability tomodulate (i.e. increase or decrease) 53BP2 binding to p53 or the53BP2-binding domain of p53, e.g., as described by Naumovski and Cleary(1996, Mol. Cell. Biol. 16:3884-3892); or for the ability to modulatethe tumor suppressive activity of p53, e.g. by any protein binding assayknown in the art, as described by Iwabuchi et al. (1994, Proc. Natl.Acad. Sci. USA 91: 6098-6102). 53BP2 has also been demonstrated toaffect the phosphorylation and dephosphorylation of p53 by 53BP2'sbinding to protein phosphatase 1 (PP1). Thus, the complexes and proteinsof the invention can be screened by assaying for changes in the level ofp53 phosphorylation (e.g., as described in Milne et al., 1994, J. Biol.Chem. 269:9253-9260) or the level of 53BP2 binding to PP1 (e.g., bymethods described supra).

β-tubulin has been shown to be upregulated in adenocarcinoma cells and,possibly, to bind proteins with Src homology 2 (SH2 domains), such asthe PDGF receptor (Shaffhausen, 1995, Biochem. Biophys. Acta1242:61-75). Thus, the complexes and proteins of the invention can bescreened by assaying for changes in β-tubulin levels (e.g., byimmunoassays with anti-β-tubulin antibodies) or for changes in β-tubulinbinding to proteins with SH2 domains.

The protein p62 associates with the p21^(waf) GTPase-activating protein(GAP), Src family tyrosine kinase SH3 domains in signalling proteins,binds RNA, interacts with ubiquitin, and also interacts with thecytosolic protein tyrosine kinase that negatively regulates the Srcfamily protein kinases. Further, p62 may also play a role in dockingcertain proteins to the cytoskeleton or membrane upon c-Src activation.Thus, the complexes and proteins of the invention can also be screenedby measuring changes in p62 binding to GAP, e.g., as described in Wanget al. (1992, Cell 69:551-558), p62 binding to proteins with SH3domains, p62 binding to RNA, e.g. as described in Wang et al. (1995, J.Biol. Chem. 270:2010-2013), interaction with ubiquitin, e.g. asdescribed by Vadlamudi et al. (1996, J. Biol. Chem 271:20235-20237), orinteraction with CSK (see Neet and Hunter, 1995, Mol. Cell. Biol.15:4908-4920). Finally, the human hnRNP G protein binds RNA; thus, thecomplexes and proteins of the invention can be screened by measuringtheir affect on the levels of hnRNP G protein binding to RNA.

The 53BP2 binding partners β-tubulin and p62 have been implicated incellular apoptosis, mRNA destabilization and ubiquitin-mediatedproteolysis associated with neurodegenerative disease. The 53BP2:53B2-IPcomplexes (particularly the 53BP2:β-tubulin and 53BP2:p62 complexes) andderivatives, analogs and fragments thereof, nucleic acids encoding the53BP2 and 53BP2-IP genes, anti-53BP2:53BP2-IP antibodies, and othermodulators of 53BP2:53BP2-IP complex activity can be tested for activityin treating or preventing neurodegenerative disease in in vitro and invivo assays.

In one embodiment, a Therapeutic of the invention can be assayed foractivity in treating or preventing neurodegenerative disease bycontacting cultured cells that exhibit an indicator of aneurodegenerative disease, for example but not limited to,hypersecretion of B-A4 peptide (Nakajima et al., 1985, Proc. Natl. Acad.Sci. USA 82:6325-6329) in vitro with the Therapeutic; and comparing thelevel of said indicator in the cells contacted with the Therapeutic,with said level of said indicator in cells not so contacted, wherein alower level in said contacted cells indicates that the Therapeutic hasactivity in treating or preventing neurodegenerative disease. Specificexamples of such cultured models for neurodegenerative disease include,but are not limited to: cultured rat endothelial cells from affected andnonaffected individuals (Maneiro et al., 1997, Methods Find. Exp. Clin.Pharmacol., 19:5-12); P19 murine embryonal carcinoma cells (Hung et al.,1992, Proc Natl Acad Sci USA 1992, 89:9439-9443); and dissociated cellcultures of cholinergic neurons from nucleus basalis of Meynert(Nakajima et al., 1985, Proc Natl Acad Sci USA, 82:6325-6329).

In another embodiment, a Therapeutic of the invention can be assayed foractivity in treating or preventing neurodegenerative disease byadministering the Therapeutic to a test animal that exhibits symptom ofa neurodegenerative disease, such as, but not limited to, cognitiviedysfunction in behavior maze test, or that is predisposed to developsymptoms or a neurodegenerative disease; and measuring the change insaid symptoms of the neurodegenerative disease after administration ofsaid Therapeutic, wherein a reduction in the severity of the symptoms ofthe neurodegenerative or prevention of the symptoms of theneurodegenerative disease indicates that the Therapeutic has activity intreating or preventing neurodegenerative disease. Such a test animal canbe any one of a number of animal models known in the art forneurodegenerative disease. These models, including those for Alzheimer'sDisease and mental retardation of trisomy 21 accurately mimic naturalhuman autoimmune diseases (Farine, 1997, Toxicol. 119:29-35). Examplesof specific models include but are not limited to: the partial trisomy16 mouse (Holtzman et al., 1996, Proc. Natl. Acad. Sci. USA93:13333-13338); bilateral nucleus basalis magnocellularis-lesioned rats(Popovic et al., 1996, Int. J. Neurosci. 86:281-299); the aged rat(Muir, 1997, Pharmacol. Biochem. Behav. 56:687-696); the PDAPPtransgenic mouse model of Alzheimer disease (Johnson-Wood et al., 1997,Proc. Natl. Acad. Sci. USA 94:1550-1555); and experimental autoimmunedementia (Oron et al., 1997, J. Neural Transm. Suppl. 49:77-84).

The 53BP2 binding partner hnRNPG has been implicated in autoimmunedisease. Accordingly, 53BP2:53BP2-IP complexes, particularly 53BP2:hnRNPG complexes, and derivatives, analogs, and fragments thereof, nucleicacids encoding the 53BP2 and 53BP2-IP genes, anti-53BP2:53BP2-IPantibodies, and other modulators of the 53BP2:53BP2-IP complex activitycan be tested for activity in treating or preventing neurodegenerativedisease in in vitro and in vivo assays.

In one embodiment, a Therapeutic of the invention can be assayed foractivity in treating or preventing autoimmune disease by contactingcultured cells that exhibit an indicator of an autoimmune reaction invitro, such as but not limited to, secretion of chemokines (Kunkel etal., 1996, J. Leukoc. Biol. 59:6-12) with the Therapeutic, and comparingthe level of said indicator in the cells contacted with the Therapeuticwith said level of said indicator in cells not so contacted, wherein alower level in said contacted cells indicates that the Therapeutic hasactivity in treating or preventing autoimmune disease. Cell models thatcan be used for such assays include, but are not limited to: leukocyteand other synovial cells that secrete chemokines mediating inflammation(Kunkel et al., 1996, J. Leukoc. Biol. 59:6-12); cerebrospinal fluidcells from animal models of multiple sclerosis (Norga et al., 1995,Inflamm. Res. 44:529-534); macrophages in experimentalautoimmunoneuritis (model of Guillain-Barre Disease (Bai et al., 1997,J. Neuroimmunol. 76:177-184); CD40/CD40L assays in monocytes (Laman etal., 1996, Crit. Rev. Immunol. 16:59-108); lymphocyte cultures for lprmice (Nagata, 1996, Prog. Mol. Subcell. Biol. 16:87-103); and culturedthyrocytes in spontaneous murine autoimmune thyroiditis (Green et al.,1996, Endocrinology 137:2823-2832).

In another embodiment, a Therapeutic of the invention can be assayed foractivity in treating or preventing autoimmune disease by administeringsaid Therapeutic to a test animal exhibiting an autoimmune reaction orwhich test animal does not exhibit an autoimmune reaction and issubsequently challenged with an agent that elicits an autoimmunereaction; and measuring the change in the autoimmune reaction after theadministration of said Therapeutic, wherein a reduction in saidautoimmune reaction or a prevention of said autoimmune reactionindicates that the Therapeutic has activity in treating or preventing anautoimmune disease.

A number of animal models of autoimmune disease are known in the art.These models, including those for arthritis, systemic lupuserythematosus, diabetes, thyroiditis, encephalitis etc., accuratelymimic natural human autoimmune diseases (Farine, 1997, Toxicol.119:29-35). Examples of specific models include but are not limited to:experimental allergic encephalomyelitis for multiple sclerosis (Brabb etal., 1997, J. Immunol. 159:497-507); thyroglobulin-induced experimentalthyroiditis (Bhatia et al., 1996 et al., 1996 213:294-300); multipleorgan-localized autoimmune disease, e.g., thyroiditis and gastritis inBALB/c nu/nu mice receiving rat thymus grafts under their renal capsules(Taguchi and Takahashi, 1996, Immunology 89:13-19); virus-inducedautoimmune diseases such as insulin-dependent diabetes mellitus(Oldstone and von Herath, 1996), experimental autoimmuneencephalomyelitis (Encinas et al., 1996, J. Neurosci. Res. 45:655-669);experimental autoimmune labyrinthitis; Freund's-adjuvant inducedrheumatoid arthritis and inbred mouse strains that develop systemiclupus erythematosus, rheumatoid arthritis, graft-vs-host disease, anddiabetes (Humphryes-Beher, 1996, Adv. Dent. Res. 10:73-75); autoimmunehepatitis (Meyer zum Buschenfelde and Dienes, 1996, Virchows Arch.429:1-12).

Screening for Antagonists and Agonists of 53BP2:53BP2-IP Complex and53BP2-IP1, 53BP2-IP2, and 53BP2-IP3

53BP2:53BP2-IP complexes, 53BP2-IP1, 53BP2-IP2, 53BP2-IP3 andderivatives, fragments and analogs thereof, as well as nucleic acidsencoding 53BP2 and 53BP2-IPs and 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 aswell as derivatives, fragments and analogs thereof, can be used toscreen for compounds that bind to 53BP2:53BP2-IP and 53BP2-IP1,53BP2-IP2, and 53BP2-IP3 nucleic acids, proteins, or derivatives andthus have potential use as agonists or antagonists of 53BP2:53BP2-IPcomplex or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 protein function. Theinvention thus provides assays to detect molecules that specificallybind to 53BP2 and 53BP2-IP, and 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3nucleic acids, proteins or derivatives. For example, recombinant cellsexpressing both 53BP2 and 53BP2-IP nucleic acids or 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 nucleic acids can be used to recombinantlyproduce the complexes or proteins in these assays, to screen formolecules that bind or interfere with 53BP2:53BP2-IP complexes or53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 function. In preferred embodiments,polypeptide analogs that have superior stabilities (but retain theability to form 53BP2:53BP2-IP complexes), e.g. 53BP2 and 53BP2-IPsmodified to be resistant to proteolytic degradation in the binding assaybuffers, or to be resistant to oxidative degradation are used to screenfor modulators (e.g. molecules generated by substitution of amino acidsat proteolytic cleavage sites, the use of chemically derivatized aminoacids at proteolytic susceptible sites, and replacement of amino acidresidues subject to oxidation, i.e. methionine and cysteine).

Molecules (e.g. putative binding partners of a 53BP2:53BP2-IP complex orof 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3) are contacted with the53BP2:53BP2-IP complex or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 proteins(or fragment thereof) under conditions conducive to binding, and thenmolecules that specifically bind to 53BP2:53BP2-IP complexes or53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 proteins are identified. Similarmethods can be used to screen for molecules that bind to 53BP2:53BP2-IPor 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 nucleic acids or derivatives.

A particular aspect of the invention relates to identifying moleculesthat inhibit or promote formation or degradation of a 53BP2:53BP2-IPcomplex, e.g. using the method described for screening inhibitors usingthe modified yeast two hybrid assay described in Section 5.7.1., infraand in U.S. patent application Ser. No. 08/663,824, filed Jun. 14, 1996,and Ser. No. 08/874,825, filed Jun. 13, 1997, both entitled“Identification and Comparison of Protein—Protein Interactions thatOccur in Populations and Identification of Inhibitors of TheseInteractions”, and both by Nandabalan et al., which are incorporated byreference herein in their entireties.

In one embodiment of the invention, a molecule that modulates activityof 53BP2 or a protein selected from the group consisting of β-tubulin,p62, hnRNP G, 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 or a complex of 53BP2and said protein is identified by contacting one or more candidatemolecules with 53BP2 in the presence of said protein; and measuring theamount of complex that forms between 53BP2 and said protein; wherein anincrease or decrease in the amount of complex that forms relative to theamount that forms in the absence of the candidate molecules indicatesthat the molecules modulate the activity of 53BP2 or said protein orsaid complex of 53BP2 and said protein. In preferred embodiments, themodulators are identified by administering the candidate molecules to atransgenic non-human animal expressing both 53BP2 and a 53BP2-IP frompromoters that are not the native 53BP2 or the native 53BP2-IPpromoters, more preferably where the candidate molecules are alsorecombinantly expressed in the transgenic non-human animal.Alternatively, the method for identifying such modulators can be carriedout in vitro, preferably with purified 53BP2, purified 53BP2-IP, andpurified candidate molecules.

Methods that can be used to carry out the foregoing are commonly knownin the art. Agents to be screened can be provided as mixtures of alimited number of specified compounds, or as compound libraries, peptidelibraries and the like. Agents to be screened may also include all formsof antisera, antisense nucleic acids, etc. that can modulate53BP2:53BP2-IP complex activity or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3activity.

By way of example, diversity libraries, such as random or combinatorialpeptide or nonpeptide libraries can be screened for molecules thatspecifically bind to a 53BP2:53BP2-IP complex or 53BP2-IP1, 53BP2-IP2,or 53BP2-IP3 protein. Many libraries are known in the art that can beused, e.g., chemically synthesized libraries, recombinant (e.g., phagedisplay libraries), and in vitro translation-based libraries.

Examples of chemically synthesized libraries are described in Fodor etal., 1991, Science 251:767-773; Houghten et al., 1991, Nature 354:84-86;Lam et al., 1991, Nature 354:82-84; Medynski, 1994, Bio/Technology12:709-710; Gallop et al., 1994, J. Medicinal Chemistry 37(9):1233-1251;Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb etal., 1994, Proc. Natl. Acad. Sci. USA 91:11422-11426; Houghten et al.,1992, Biotechniques 13:412; Jayawickreme et al., 1994, Proc. Natl. Acad.Sci. USA 91:1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA90:11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lerner,1992, Proc. Natl. Acad. Sci. USA 89:5381-5383.

Examples of phage display libraries are described in Scott and Smith,1990, Science 249:386-390; Devlin et al., 1990, Science, 249:404-406;Christian, R. B., et al., 1992, J. Mol. Biol. 227:711-718); Lenstra,1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65;and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.

In vitro translation-based libraries include but are not limited tothose described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991;and Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA 91:9022-9026.

By way of examples of nonpeptide libraries, a benzodiazepine library(see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712)can be adapted for use. Peptoid libraries (Simon et al., 1992, Proc.Natl. Acad. Sci. USA 89:9367-9371) can also be used. Another example ofa library that can be used, in which the amide functionalities inpeptides have been permethylated to generate a chemically transformedcombinatorial library, is described by Ostresh et al. (1994, Proc. Natl.Acad. Sci. USA 91:11138-11142).

Screening the libraries can be accomplished by any of a variety ofcommonly known methods. See, e.g., the following references, whichdisclose screening of peptide libraries: Parmley and Smith, 1989, Adv.Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390;Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg et al., 1992,Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al., 1992,Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA89:6988-6992; Ellington et al., 1992, Nature 355:850-852; U.S. Pat. Nos.5,096,815, 5,223,409, and 5,198,346, all to Ladner et al.; Rebar andPabo, 1993, Science 263:671-673; and PCT Publication No. WO 94/18318.

In a specific embodiment, screening can be carried out by contacting thelibrary members with a 53BP2:53BP2-IP complex or a 53BP2-IP1, 53BP2-IP2,or 53BP2-IP3 protein (or nucleic acid or derivative) immobilized on asolid phase and harvesting those library members that bind to theprotein (or nucleic acid or derivative). Examples of such screeningmethods, termed “panning” techniques are described by way of example inParmley and Smith, 1988, Gene 73:305-318; Fowlkes et al., 1992,BioTechniques 13:422-427; PCT Publication No. WO 94/18318; and inreferences cited hereinabove.

In another specific embodiment, fragments and/or analogs of 53BP2 or a53BP2-IP, especially peptidomimetics, are screened for activity ascompetitive or non-competitive inhibitors of 53BP2:53BP2-IP complexformation, and thereby inhibit 53BP2-IP complex activity.

In a preferred embodiment, molecules that bind to 53BP2:53BP2-IPcomplexes and 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 proteins can bescreened for using the modified yeast two hybrid system described inSection 5.7.1 and exemplified in Section 6.1, infra.

In one embodiment, agents that modulate (i.e. inhibit, antagonize, oragonize) 53BP2:53BP2-IP complex activity can be screened using a bindinginhibition assay, wherein agents are screened for their ability toinhibit formation of a 53BP2:53BP2-IP complex under aqueous, orphysiological, binding conditions in which 53BP2:53BP2-IP complexformation occurs in the absence of the agent to be tested. Agents thatinterfere with the formation of 53BP2:53BP2-IP complexes are identifiedas antagonists of complex formation.

Methods for screening may involve labeling the complex proteins withradioligands (e.g. ¹²⁵I or 3H), magnetic ligands (e.g. paramagneticbeads covalently attached to photobiotin acetate), and florescentligands (e.g. fluorescein or rhodamine) or enzyme ligands (e.g.luciferase or beta-galactosidase). The reactants that bind in solutioncan then be isolated by one of many techniques known in the art,including but not restricted to, co-immunoprecipitation of the labeledmoiety using antisera against the unlabeled binding partner (or labeledbinding partner with a distinguishable marker from that used on thelabeled moiety) protein, immunoaffinity chromatography, size exclusionchromatography, and gradient density centrifugation. In a preferredembodiment, one binding partner is a small fragment or peptidomimeticthat is not retained by a commercially available filter. Upon binding,the labeled species is then unable to pass through the filter, providingfor a simple assay of complex formation.

Methods commonly known in the art are used to label at least one of themembers of the 53BP2:53BP2-IP complex. Suitable labeling includes, butis not limited to, radiolabeling by incorporation of radiolabeled aminoacids, e.g. ³H-leucine of ³⁵S-methionine, radiolabeling bypost-translational iodination with ¹²⁵I or ¹³¹I using the chloramine Tmethod, Bolton-Hunter reagents, etc., or labeling with ³²P usingphosphorylase and inorganic radiolabeled phosphorous, biotin labelingwith photobiotin-acetate and sunlamp exposure, etc. In cases where oneof the members of the 53BP2:53BP2-IP complex is immobilized, e.g. asdescribed infra, the free species is labeled. Where neither of theinteracting species is immobilized, each can be labelled with adistinguishable marker such that isolation of both moieties can befollowed to provide for more accurate quantitation, and to distinguishthe formation of homomeric from heteromeric complexes. Methods thatutilize accessory proteins that bind to one of the modified interactantsto improve the sensitivity of detection, increase the stability of thecomplex, etc. are provided.

Typical binding conditions are, for example, but not by way oflimitation, in an aqueous salt solution of 10-250 mM NaCl, 5-50 mMTris-HCl, pH 5-8, 0.5% Triton X-100 or other detergent that improvesspecificity of interaction. Metal chelators and/or divalent cations maybe added to improve binding and/or reduce proteolysis. Reactiontemperatures may include 4, 10, 15, 22, 25, 35, or 42 degrees Celsius,and time of incubation is typically at least 15 seconds, but longertimes are preferred to allow binding equilibrium to occur. Particular53BP2:53BP2-IP complexes can be assayed using routine protein bindingassays to determine optimal binding conditions for reproducible binding.

The physical parameters of complex formation can be analyzed byquantitation of complex formation using assay methods specific for thelabel used, e.g. liquid scintillation counting for radioactivitydetection, enzyme activity measurements for enzyme label, etc. Thereaction results are then analyzed utilizing Scatchard analysis, Hillanalysis, and other methods commonly known in the arts (see, e.g.,Proteins, Structures, and Molecular Principles, (1984) Creighton, ed.,W. H. Freeman and Company, New York).

In a second common approach to binding assays, one of the bindingspecies is immobilized on a filter, in a microtiter plate well, in atest tube, to a chromatography matrix, etc., either covalently ornon-covalently. Proteins can be covalently immobilized using any methodwell known in the art, for example, but not limited to the method ofKadonaga and Tjian (1986, Proc. Natl. Acad. Sci. USA 83: 5889-5893,1986), i.e., linkage to a cyanogen-bromide derivatized substrate such asCNBr-Sepahrose 4B. Where needed, the use of spacers can reduce sterichindrance from the substrate. Non-covalent attachment of proteins to asubstrate include, but are not limited to, attachment of a protein to acharged surface, binding with specific antibodies, binding to a thirdunrelated IP, etc.

In one embodiment, immobilized 53BP2 is used to assay for binding with aradioactively-labeled 53BP2-IP in the presence and absence of a compoundto be tested for its ability to modulate 53BP2:53BP2-IP complexformation. The binding partners are allowed to bind under aqueous, orphysiological, conditions (e.g. the conditions under which the originalinteraction was detected). Conversely, in another embodiment, the53BP2-IP is immobilized and contacted with the labeled 53BP2 protein orderivative thereof under binding conditions.

Assays of agents (including cell extracts or library pool) forcompetition for binding of one member of a 53BP2:53BP2-IP complex (orderivatives thereof) with the other member of the 53BP2:53BP2-IP complex(labeled by any means, e.g. those means described supra), are providedto screen for competitors of 53BP2:53BP2-IP complex formation.

In specific embodiments, blocking agents to inhibit non-specific bindingof reagents to other protein components, or absorptive losses ofreagents to plastics, immobilization matrices, etc., are included in theassay mixture. Blocking agents include, but are not restricted to bovineserum albumin, beta-casein, nonfat dried milk, Denhardt's reagent,Ficoll, polyvinylpyrolidine, nonionic detergents(NP40, Triton X-100,Tween 20, Tween 80, etc.), ionic detergents (e.g. SDS, LDS, etc.),polyethyleneglycol, etc. Appropriate blocking agent concentrations allow53BP2:53BP2-IP complex formation.

After binding is performed, unbound, labelled protein is removed in thesupernatant, and the immobilized protein with any bound, labelledprotein is washed extensively. The amount of label bound is thenquantitated using standard methods in the art to detect the label asdescribed supra.

Assays for Proteins-Protein Interactions

One aspect of the present invention provides methods for assaying andscreening fragments, derivatives and analogs of derivatives, analogs andfragments of 53BP2-interacting proteins (for binding to 53BP2 peptides).Derivatives, analogs and fragments of 53BP2-IPs that interact with 53BP2can be identified by means of a yeast two hybrid assay system (Fieldsand Song, 1989, Nature 340: 245-246; U.S. Pat. No. 5,283,173 by Fieldsand Song) or, more preferably, an improvement thereof as described inU.S. patent application Ser. No. 08/663,824, filed Jun. 14, 1996, andSer. No. 08/874,825, filed Jun. 13, 1997, both entitled “Identificationand Comparison of Protein-Protein Interactions that Occur in Populationsand Identification of Inhibitors of These Interactions”, and both byNandabalan et al., which are incorporated by reference herein in theirentireties. Because the interactions are screened for in yeast, theintermolecular protein interactions detected in this system generallyoccur under physiological conditions that mimic the conditions inmammalian cells (Chien et al., 1991, Proc. Natl. Acad. Sci. USA 88:9578-9581.)

Identification of interacting proteins by the improved yeast two hybridsystem is based upon the detection of the expression of a reporter gene(“Reporter Gene”), the transcription of which is dependent upon thereconstitution of a transcriptional regulator by the interaction of twoproteins, each fused to one half of the transcriptional regulator. Thebait (53BP2 or derivative or analog) and prey (proteins to be tested forability to interact with the bait) proteins are expressed as fusionproteins to a DNA binding domain, and to a transcriptional regulatorydomain, respectively, or vice versa. In various specific embodiments,the prey has a complexity of at least 50, 100, 500, 1,000, 5,000,10,000, or 50,000; or has a complexity in the range of 25 to 100,000,100 to 100,000, 50,000 to 100,000, or 10,000 to 500,000. For example,the prey population can be one or more nucleic acids encoding mutants ofa 53BP2-IP (e.g., as generated by site-directed mutagenisis or anothermethod of making mutations in a nucleotide sequence). Preferably, theprey populations are proteins encoded by DNA, e.g., cDNA or genomic DNAor synthetically generated DNA. For example, the populations can beexpressed from chimeric genes comprising cDNA sequences from anun-characterized sample of a population of cDNA from mammalian RNA.Preferably, the prey population are proteins encoded by DNA, e.g., cDNAor genomic DNA or synthetically generated DNA.

In a specific embodiment, recombinant biological libraries expressingrandom peptides can be used as the source of prey nucleic acids.

In another embodiment, the invention provides methods for screening forinhibitors of the interacting proteins identified herein. Briefly, theprotein-protein interaction assay can be carried out as describedherein, except that it is done in the presence of one or more candidatemolecules. An increase or decrease in Reporter Gene activity relative tothat present when the one or more candidate molecules are absentindicates that the candidate molecule has an effect on the interactingpair. In a preferred method, inhibition of the interaction is selectedfor (i.e. inhibition of the interaction is necessary for the cells tosurvive), for example, where the interaction activates the URA3 gene,causing yeast to die in medium containing the chemical 5-fluorooroticacid (Rothstein, 1983, Meth. Enzymol. 101:167-180). The identificationof inhibitors of such interactions can also be accomplished, forexample, but not by way of limitation, using competitive inhibitorassays, as described supra.

In general, proteins of the bait and prey populations are provided asfusion (chimeric) proteins (preferably by recombinant expression of achimeric coding sequence) containing each protein contiguous to apre-selected sequence. For one population, the pre-selected sequence isa DNA binding domain. The DNA binding domain can be any DNA bindingdomain, as long as it specifically recognizes a DNA sequence within apromoter. For example, the DNA binding domain is of a transcriptionalactivator or inhibitor. For the other population, the pre-selectedsequence is an activator or inhibitor domain of a transcriptionalactivator or inhibitor, respectively. The regulatory domain alone (notas a fusion to a protein sequence) and the DNA-binding domain alone (notas a fusion to a protein sequence) preferably do not detectably interact(so as to avoid false positives in the assay). The assay system furtherincludes a reporter gene operably linked to a promoter that contains abinding site for the DNA binding domain of the transcriptional activator(or inhibitor). Accordingly, in the method of the invention, binding ofa 53BP2 fusion protein to a prey fusion protein leads to reconstitutionof a transcriptional activator (or inhibitor) which activates (orinhibits) expression of the Reporter Gene. The activation oftranscription of the Reporter Gene occurs intracellularly, e.g., inprokaryotic or eukaryotic cells, preferably in cell culture.

The promoter that is operably linked to the reporter gene nucleotidesequence can be a native or non-native promoter of the nucleotidesequence, and the DNA binding site(s) that are recognized by the DNAbinding domain portion of the fusion protein can be native to thepromoter (if the promoter normally contains such binding site(s)) ornon-native. Thus, for example, one or more tandem copies (e.g., 4 or 5copies) of the appropriate DNA binding site can be introduced upstreamof the TATA box in the desired promoter (e.g., in the area of position−100 to −400). In a preferred aspect, 4 or 5 tandem copies of the 17 bpUAS (GAL4 DNA binding site) are introduced upstream of the TATA box inthe desired promoter, which is upstream of the desired coding sequencefor a selectable or detectable marker. In a preferred embodiment, theGALl-10 promoter is operably fused to the desired nucleotide sequence;the GALl-10 promoter already contains 5 binding sites for GAL4.Alternatively, the transcriptional activation binding site of thedesired gene(s) can be deleted and replaced with GAL4 binding sites(Bartel et al., 1993, BioTechniques 14(6):920-924; Chasman et al., 1989,Mol. Cell. Biol. 9:4746-4749). The Reporter Gene preferably contains thesequence encoding a detectable or selectable marker the expression ofwhich is regulated by the transcriptional activator, such that themarker is either turned on or off in the cell in response to thepresence of a specific interaction. Preferably, the assay is carried outin the absence of background levels of the transcriptional activator(e.g., in a cell that is mutant or otherwise lacking in thetranscriptional activator). In one embodiment, more than one ReporterGene is used to detect transcriptional activation, e.g., one ReporterGene encoding a detectable marker and one or more Reporter Genesencoding different selectable markers. The detectable marker can be anymolecule that can give rise to a detectable signal, e.g., a fluorescentprotein or a protein that can be readily visualized or that isrecognizable by a specific antibody. The selectable marker can be anyprotein molecule that confers ability to grow under conditions that donot support the growth of cells not expressing the selectable marker,e.g., the selectable marker is an enzyme that provides an essentialnutrient and the cell in which the interaction assay occurs is deficientin the enzyme and the selection medium lacks such nutrient. The ReporterGene can either be under the control of the native promoter thatnaturally contains a binding site for the DNA binding protein, or underthe control of a heterologous or synthetic promoter.

The activation domain and DNA binding domain used in the assay can befrom a wide variety of transcriptional activator proteins, as long asthese transcriptional activators have separable binding andtranscriptional activation domains. For example, the GAL4 protein of S.cerevisiae, the GCN4 protein of S. cerevisiae (Hope and Struhl, 1986,Cell 46:885-894), the ARD1 protein of S. cerevisiae (Thukral et al.,1989, Mol. Cell. Biol. 9:2360-2369), and the human estrogen receptor(Kumar et al., 1987, Cell 51:941-951) have separable DNA binding andactivation domains. The DNA binding domain and activation domain thatare employed in the fusion proteins need not be from the sametranscriptional activator. In a specific embodiment, a GAL4 or LEXA DNAbinding domain is employed. In another specific embodiment, a GAL4 orherpes simplex virus VP16 (Triezenberg et al., 1988, Genes Dev.2:730-742) activation domain is employed. In a specific embodiment,amino acids 1-147 of GAL4 (Ma et al., 1987, Cell 48:847-853; Ptashne etal., 1990, Nature 346:329-331) is the DNA binding domain, and aminoacids 411-455 of VP16 (Triezenberg et al., 1988, Genes Dev. 2:730-742;Cress et al., 1991, Science 251:87-90) is the activation domain.

In a preferred embodiment, the yeast transcription factor GAL4 isreconstituted by the protein-protein interaction and the host strain ismutant for GAL4. In another embodiment, the DNA-binding domain is Ace1Nand/or the activation domain is Ace1, the DNA binding and activationdomains of the Ace1 protein, respectively. Ace1 is a yeast protein thatactivates transcription from the CUP1 operon in the presence of divalentcopper. CUP1 encodes metallothionein, which chelates copper, and theexpression of CUP1 protein allows growth in the presence of copper,which is otherwise toxic to the host cells. The Reporter Gene can alsobe a CUP1-lacZ fusion that expresses the enzyme β-galactosidase(detectable by routine chromogenic assay) upon binding of areconstituted Ace1N transcriptional activator (see Chaudhuri et al.,1995, FEBS Letters 357:221-226). In another specific embodiment, the DNAbinding domain of the human estrogen receptor is used, with a ReporterGene driven by one or three estrogen receptor response elements (LeDouarin et al., 1995, Nucl. Acids. Res. 23:876-878).

The DNA binding domain and the transcription activator/inhibitor domaineach preferably has a nuclear localization signal (see Ylikomi et al.,1992, EMBO J. 11:3681-3694; Dingwall and Laskey, 1991, TIBS 16:479-481)functional in the cell in which the fusion proteins are to be expressed.

To facilitate isolation of the encoded proteins, the fusion constructscan further contain sequences encoding affinity tags such asglutathione-S-transferase or maltose-binding protein or an epitope of anavailable antibody, for affinity purification (e.g., binding toglutathione, maltose, or a particular antibody specific for the epitope,respectively) (Allen et al., 1995, TIBS 20:511-516). In anotherembodiment, the fusion constructs further comprise bacterial promotersequences for recombinant production of the fusion protein in bacterialcells (see Allen et al., 1995, TIBS 20:511-516).

The host cell in which the interaction assay occurs can be any cell,prokaryotic or eukaryotic, in which transcription of the Reporter Genecan occur and be detected, including but not limited to mammalian (e.g.,monkey, chicken, mouse, rat, human, bovine), bacteria, and insect cells,and is preferably a yeast cell. Expression constructs encoding andcapable of expressing the binding domain fusion proteins, thetranscriptional activation domain fusion proteins, and the Reporter Geneproduct(s) are provided within the host cell, by mating of cellscontaining the expression constructs, or by cell fusion, transformation,electroporation, microinjection, etc. In a specific embodiment in whichthe assay is carried out in mammalian cells (e.g., hamster cells), theDNA binding domain is the GAL4 DNA binding domain, the activation domainis the herpes simplex virus VP16 transcriptional activation domain, andthe Reporter Gene contains the desired coding sequence operably linkedto a minimal promoter element from the adenovirus E1B gene driven byseveral GAL4 DNA binding sites (see Fearon et al., 1992, Proc. Natl.Acad. Sci. USA 89:7958-7962). The host cell used should not express anendogenous transcription factor that binds to the same DNA site as thatrecognized by the DNA binding domain fusion population. Also,preferably, the host cell is mutant or otherwise lacking in anendogenous, functional form of the Reporter Gene(s) used in the assay.

Various vectors and host strains for expression of the two fusionprotein populations in yeast are known and can be used (see, e.g.,Fields et al., U.S. Pat. No. 5,1468,614 dated Nov. 21, 1995; Bartel etal., 1993, “Using the two-hybrid system to detect protein-proteininteractions,” in Cellular Interactions in Development, Hartley, D. A.(ed.), Practical Approach Series xviii, IRL Press at Oxford UniversityPress, New York, N.Y., pp. 153-179; Fields and Sternglanz, 1994, TIG10:286-292). By way of example but not limitation, yeast strains orderivative strains made therefrom, which can be used are N105, N106,N1051, N1061, and YULH, as described in Section 6.3, infra. Exemplarystrains that can be used in the assay of the invention also include, butare not limited to, the following: Y190: MATa, ura3-52, his3-200,lys2-801, ade2-101, trp1-901, leu2-3,112, gal4Δ, gal80Δ, cyh^(r)2,LYS2::GALl_(UAS)-HIS3_(TATA)HIS3, URA3::GALl_(UAS)-GALl_(TATA)-lacZ(available from Clontech, Palo Alto, Calif.; Harper et al., 1993, Cell75:805-816). Y190 contains HIS3 and lacZ Reporter Genes driven by GAL4binding sites. CG-1945: MATa, ura3-52, his3-200, lys2-801, ade2-101,trp1-901, leu2-3,112, gal4-542, gal80-538, cyhr^(r)2,LYS2::GALl_(UAS)-HIS3_(TATA)HIS3,URA3::GALl_(UAS17mers(×3))-CYC1_(TATA)-laCZ (available from Clontech).CG-1945 contains HIS3 and lacZ Reporter Genes driven by GAL4 bindingsites. Y187: MAT-α, ura3-52, his3-200, ade2-101, trp1-901, leu2-3,112,gal4Δ, gal80Δ, URA3:: GAL1_(UAS)-GAL1_(TATA)-lacZ (available fromClontech). Y187 contains a lacZ Reporter Gene driven by GAL4 bindingsites. SFY526: MATa, ura3-52, his3-200, lys2-801, ade2-101, trp1-901,leu2-3,112, gal4-542, gal80-538, can^(r),URA3::GAL1-lacZ (available fromClontech). SFY526 contains HIS3 and lacZ Reporter Genes driven by GAL4binding sites. HF7c: MATa, ura3-52, his3-200, lys2-801, ade2-101,trp1-901, leu2-3,112, gal4-542, gal80-538, LYS2::GAL1-HIS3.URA3::GAL1_(UAS 17 MERS (×3))-CYC1-lacZ (available from Clontech). HF7ccontains HIS3 and lacZ Reporter Genes driven by GAL4 binding sites.YRG-2: MATa, ura3-52, his3-200, lys2-801, ade2-101, trp1-901,leu2-3,112, gal4-542, gal80-538 LYS2::GAL1_(UAS)-GAL1_(TATA)-HIS3,URA3::GAL1_(UAS 17mers (×3))-CYC1-lacZ (available from Stratagene).YRG-2 contains HIS3 and lacZ Reporter Genes driven by GAL4 bindingsites.

Many other strains commonly known and available in the art can be used.

If not already lacking in endogenous Reporter Gene activity, cellsmutant in the Reporter Gene may be selected by known methods, or thecells can be made mutant in the target Reporter Gene by knowngene-disruption methods prior to introducing the Reporter Gene(Rothstein, 1983, Meth. Enzymol. 101:202-211).

In a specific embodiment, plasmids encoding the different fusion proteinpopulations can be both introduced into a single host cell (e.g., ahaploid yeast cell) containing one or more Reporter Genes, byco-transformation, to conduct the assay for protein-proteininteractions. Or, preferably, the two fusion protein populations areintroduced into a single cell either by mating (e.g. for yeast cells) orcell fusions (e.g., of mammalian cells). In a mating type assay,conjugation of haploid yeast cells of opposite mating type that havebeen transformed with a binding domain fusion expression construct(preferably a plasmid) and an activation (or inhibitor) domain fusionexpression construct (preferably a plasmid), respectively, delivers bothconstructs into the same diploid cell. The mating type of a yeast strainmay be manipulated by transformation with the HO gene (Herskowitz andJensen, 1991, Meth. Enzymol. 194:132-146).

In a preferred embodiment, a yeast interaction mating assay is employed,using two different types of host cells, strain-types a and alpha, ofthe yeast Saccharomyces cerevisiae. The host cell preferably contains atleast two Reporter Genes, each with one or more binding sites for theDNA-binding domain (e.g., of a transcriptional activator). The activatordomain and DNA binding domain are each parts of chimeric proteins formedfrom the two respective populations of proteins. One set of host cells,for example the a strain cells, contains fusions of the library ofnucleotide sequences with the DNA-binding domain of a transcriptionalactivator, such as GAL4. The hybrid proteins expressed in this set ofhost cells are capable of recognizing the DNA-binding site on theReporter Gene. The second set of yeast host cells, for example alphastrain cells, contains nucleotide sequences encoding fusions of alibrary of DNA sequences fused to the activation domain of atranscriptional activator. In a preferred embodiment, the fusion proteinconstructs are introduced into the host cell as a set of plasmids. Theseplasmids are preferably capable of autonomous replication in a hostyeast cell and preferably can also be propagated in E. coli. The plasmidcontains a promoter directing the transcription of the DNA binding oractivation domain fusion genes, and a transcriptional terminationsignal. The plasmid also preferably contains a selectable marker gene,permitting selection of cells containing the plasmid. The plasmid can besingle-copy or multi-copy. Single-copy yeast plasmids that have theyeast centromere may also be used to express the activation and DNAbinding domain fusions (Elledge et al., 1988, Gene 70:303-312). Inanother embodiment, the fusion constructs are introduced directly intothe yeast chromosome via homologous recombination. The homologousrecombination for these purposes is mediated through yeast sequencesthat are not essential for vegetative growth of yeast, e.g., MER2, MER1,ZIP1, REC102, or ME14 gene.

Bacteriophage vectors can also be used to express the DNA binding domainand/or activation domain fusion proteins. Libraries can generally beprepared faster and more easily from bacteriophage vectors than fromplasmid vectors.

In a specific embodiment, the invention provides a method of detectingone or more protein-protein interactions comprising (a) recombinantlyexpressing 53BP2 or a derivative or analog thereof in a first populationof yeast cells being of a first mating type and comprising a firstfusion protein containing the 53BP2 sequence and a DNA binding domain,wherein said first population of yeast cells contains a first nucleotidesequence operably linked to a promoter driven by one or more DNA bindingsites recognized by said DNA binding domain such that an interaction ofsaid first fusion protein with a second fusion protein, said secondfusion protein comprising a transcriptional activation domain, resultsin increased transcription of said first nucleotide sequence; (b)negatively selecting to eliminate those yeast cells in said firstpopulation in which said increased transcription of said firstnucleotide sequence occurs in the absence of said second fusion protein;(c) recombinantly expressing in a second population of yeast cells of asecond mating type different from said first mating type, a plurality ofsaid second fusion proteins, each second fusion protein comprising asequence of a fragment, derivative or analog of a 53BP2-IP and anactivation domain of a transcriptional activator, in which theactivation domain is the same in each said second fusion protein; (d)mating said first population of yeast cells with said second populationof yeast cells to form a third population of diploid yeast cells,wherein said third population of diploid yeast cells contains a secondnucleotide sequence operably linked to a promoter driven by a DNAbinding site recognized by said DNA binding domain such that aninteraction of a first fusion protein with a second fusion proteinresults in increased transcription of said second nucleotide sequence,in which the first and second nucleotide sequences can be the same ordifferent; and (e) detecting said increased transcription of said firstand/or second nucleotide sequence, thereby detecting an interactionbetween a first fusion protein and a second fusion protein.

In a preferred embodiment, the bait 53BP2 sequence and the prey libraryof chimeric genes are combined by mating the two yeast strains on solidmedia for a period of approximately 6-8 hours. In a less preferredembodiment, the mating is performed in liquid media. The resultingdiploids contain both kinds of chimeric genes, i.e., the DNA-bindingdomain fusion and the activation domain fusion.

Preferred reporter genes include URA3, HIS3 and/or the lacZ genes (see,e.g., Rose and Botstein, 1983, Meth. Enzymol. 101:167-180) operablylinked to GAL4 DNA-binding domain recognition elements. Other reportergenes comprise the functional coding sequences for, but not limited to,Green Fluorescent Protein (GFP) (Cubitt et al., 1995, Trends Biochem.Sci. 20:448-455), luciferase, LEU2, LYS2, ADE2, TRP1, CAN1, CYH2, GUS,CUP1 or chloramphenicol acetyl transferase (CAT). Expression of LEU2,LYS2, ADE2 and TRP1 are detected by growth in a specific defined media;GUS and CAT can be monitored by well known enzyme assays; and CAN1 andCYH2 are detected by selection in the presence of canavanine andcycloheximide. With respect to GFP, the natural fluorescence of theprotein is detected.

In a specific embodiment, transcription of the Reporter Gene is detectedby a linked replication assay. For example, as described by Vasavada etal. (1991, Proc. Natl. Acad. Sci. USA 88:10686-10690), expression ofSV40 large T antigen is under the control of the ElB promoter responsiveto GAL4 binding sites. The replication of a plasmid containing the SV40origin of replication, indicates the reconstruction of the GAL4 proteinand a protein-protein interaction. Alternatively, a polyoma virusreplicon can be employed (Vasavada et al., 1991, Proc. Natl. Acad. Sci.USA 88:10686-0690).

In another embodiment, the expression of Reporter Genes that encodeproteins can be detected by immunoassay, i.e., by detecting theimmunospecific binding of an antibody to such protein, which antibodycan be labeled, or alternatively, which antibody can be incubated with alabeled binding partner to the antibody, so as to yield a detectablesignal. Alam and Cook (1990, Anal. Biochem. 188:245-254) disclosenon-limiting examples of detectable marker genes that can be operablylinked to a transcriptional regulatory region responsive to areconstituted transcriptional activator, and thus used as ReporterGenes.

The activation of Reporter Genes like URA3 or HIS3 enables the cells togrow in the absence of uracil or histidine, respectively, and henceserves as a selectable marker. Thus, after mating, the cells exhibitingprotein-protein interactions are selected by the ability to grow inmedia lacking a nutritional component, such as uracil or histidine,respectively (referred to as −URA (minus URA) and −HIS (minus HIS)medium, respectively). The −HIS medium preferably contains3-amino-1,2,4-triazole (3-AT), which is a competitive inhibitor of theHIS3 gene product and thus requires higher levels of transcription inthe selection (see Durfee et al., 1993, Genes Dev. 7:555-569).Similarly, 6-azauracil, which is an inhibitor of the URA3 gene product,can be included in −URA medium (Le Douarin et al., 1995, Nucl. AcidsRes. 23:876-878). URA3 gene activity can also be detected and/ormeasured by determining the activity of its gene product,orotidine-51-monophosphate decarboxylase (Pierrat et al., 1992, Gene119:237-245; Wolcott et al., 1966, Biochem. Biophys. Acta 122:532-534).In other embodiments of the invention, the activities of the reportergenes like lacZ or GFP are monitored by measuring a detectable signal(e.g., fluorescent or chromogenic) that results from the activation ofthese Reporter Genes. For example, lacZ transcription can be monitoredby incubation in the presence of a chromogenic substrate, such as X-gal(5-bromo-4-chloro-3-indolyl-α-D-galactoside), for its encoded enzyme,β-galactosidase. The pool of all interacting proteins isolated by thismanner from mating the 53BP2 sequence product and the library identifiesthe “53BP2 interactive population”.

In a preferred embodiment of the invention, false positives arising fromtranscriptional activation by the DNA binding domain fusion proteins inthe absence of a transcriptional activator domain fusion protein areprevented or reduced by negative selection for such activation within ahost cell containing the DNA binding fusion population, prior toexposure to the activation domain fusion population. By way of example,if such cell contains URA3 as a Reporter Gene, negative selection iscarried out by incubating the cell in the presence of 5-fluorooroticacid (5-FOA, which kills URA+ cells (Rothstein, 1983, Meth. Enzymol.101:167-180). Hence, if the DNA-binding domain fusions by themselvesactivate transcription, the metabolism of 5-FOA will lead to cell deathand the removal of self-activating DNA-binding domain hybrids.

Negative selection involving the use of a selectable marker as aReporter Gene and the presence in the cell medium of an agent toxic orgrowth inhibitory to the host cells in the absence of Reporter Genetranscription is preferred, since it allows a higher rate of processingthan other methods. As will be apparent, negative selection can also becarried out on the activation domain fusion population prior tointeraction with the DNA binding domain fusion population, by similarmethods, either alone or in addition to negative selection of the DNAbinding fusion population.

Negative selection can also be carried out on the recovered53BP2:53BP2-IP pairs by known methods (see, e.g., Bartel et al., 1993,BioTechniques 14:920-924) although pre-negative selection (prior to theinteraction assay), as described above, is preferred. For example, eachplasmid encoding a protein (peptide or polypeptide) fused to theactivation domain (one-half of a detected interacting pair) can betransformed back into the original screening strain, either alone orwith a plasmid encoding only the DNA-binding domain, the DNA-bindingdomain fused to the detected interacting protein, or the DNA-bindingdomain fused to a protein that does not affect transcription orparticipate in the protein-protein interaction; a positive interactiondetected with any plasmid other than that encoding the DNA-bindingdomain fusion to the detected interacting protein is deemed a falsepositive and eliminated from the screen.

In a preferred embodiment, the 53BP2 plasmid population is transformedin a yeast strain of a first mating type (a or alpha), and the secondplasmid population (containing the library of DNA sequences) istransformed in a yeast strain of different mating type. Both strains arepreferably mutant for URA3 and HIS3, and contain HIS3, and optionallylacZ, as a Reporter Genes. The first set of yeast cells are positivelyselected for the 53BP2 plasmids and are negatively selected for falsepositives by incubation in medium lacking the selectable marker (e.g.,tryptophan) and containing 5-FOA. Yeast cells of the second mating typeare transformed with the second plasmid population, and are positivelyselected for the presence of the plasmids containing the library offusion proteins. Selected cells are pooled. Both groups of pooled cellsare mixed together and mating is allowed to occur on a solid phase. Theresulting diploid cells are then transferred to selective media thatselects for the presence of each plasmid and for activation of ReporterGenes.

In a preferred embodiment of the invention, after an interactivepopulation is obtained, the DNA sequences encoding the pairs ofinteractive proteins are isolated by a method wherein either theDNA-binding domain hybrids or the activation domain hybrids areamplified, in separate respective reactions. Preferably, theamplification is carried out by polymerase chain reaction (PCR) (U.S.Pat. Nos. 4,683,202. 4,683,195 and 4,889,818; Gyllenstein et al., 1988,Proc. Natl. Acad. Sci. USA 85:7652-7656; Ochman et al., 1988, Genetics120:621-623; Loh et al., 1989, Science 243:217-220; Innis et al., 1990,PCR Protocols, Academic Press, Inc., San Diego, Calif.), using pairs ofoligonucleotide primers specific for either the DNA-binding domainhybrids or the activation domain hybrids. This PCR reaction can also beperformed on pooled cells expressing interacting protein pairs,preferably pooled arrays of interactants. Other amplification methodsknown in the art can be used, including but not limited to ligase chainreaction (see EP 320,308) use of Qβ replicase, or methods listed inKricka et al., 1995, Molecular Probing, Blotting, and Sequencing, Chap.1 and Table IX, Academic Press, New York.

The plasmids encoding the DNA-binding domain hybrid and the activationdomain hybrid proteins can also be isolated and cloned by any of themethods well known in the art. For example, but not by way oflimitation, if a shuttle (yeast to E. coli) vector is used to expressthe fusion proteins, the genes can be recovered by transforming theyeast DNA into E. coli and recovering the plasmids from E. coli (see,e.g., Hoffman et al., 1987, Gene 57:267-272). Alternatively, the yeastvector can be isolated, and the insert encoding the fusion proteinsubcloned into a bacterial expression vector, for growth of the plasmidin E. coli.

Pharmaceutical Compositions and Therapeutic/prophylactic Administration

The invention provides methods of treatment (and prophylaxis) byadministration to a subject of an effective amount of a Therapeutic ofthe invention. In a preferred aspect, the Therapeutic is substantiallypurified. The subject is preferably an animal, including but not limitedto animals such as cows, pigs, horses, chickens, cats, dogs, etc., andis preferably a mammal, and most preferably human. In a specificembodiment, a non-human mammal is the subject.

Formulations and methods of administration that can be employed when theTherapeutic comprises a nucleic acid are described in Sections 5.5.6 and5.5.7 above; additional appropriate formulations and routes ofadministration can be selected from among those described hereinbelow.

Various delivery systems are known and can be used to administer aTherapeutic of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe Therapeutic, receptor-mediated endocytosis (see, e.g., Wu and Wu,1987, J. Biol. Chem. 262:4429-4432), construction of a Therapeuticnucleic acid as part of a retroviral or other vector, etc. Methods ofintroduction include but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds may be administered by any convenient route,for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Inaddition, it may be desirable to introduce the pharmaceuticalcompositions of the invention into the central nervous system by anysuitable route, including intraventricular and intrathecal injection;intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an ommayareservoir. Pulmonary administration can also be employed, e.g., by useof an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. In oneembodiment, administration can be by direct injection at the site (orformer site) of a malignant tumor or neoplastic or pre-neoplastictissue.

In another embodiment, the Therapeutic can be delivered in a vesicle, inparticular a liposome (see Langer, Science 249:1527-1533 (1990); Treatet al., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

In yet another embodiment, the Therapeutic can be delivered in acontrolled release system. In one embodiment, a pump may be used (seeLanger, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem.23:61 (1983); see also Levy et al., Science 228:190 (1985); During etal., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105(1989)). In yet another embodiment, a controlled release system can beplaced in proximity of the therapeutic target, i.e., the brain, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In a specific embodiment where the Therapeutic is a nucleic acidencoding a protein Therapeutic, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid Therapeuticcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of aTherapeutic, and a pharmaceutically acceptable carrier. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the Therapeutic, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The Therapeutics of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The amount of the Therapeutic of the invention which will be effectivein the treatment of a particular disorder or condition will depend onthe nature of the disorder or condition, and can be determined bystandard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. However, suitable dosage ranges forintravenous administration are generally about 20-500 micrograms ofactive compound per kilogram body weight. Suitable dosage ranges forintranasal administration are generally about 0.01 pg/kg body weight to1 mg/kg body weight. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

Suppositories generally contain active ingredient in the range of 0.5%to 10% by weight; oral formulations preferably contain 10% to 95% activeingredient.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Animal Models

The invention also provides animal models. In one embodiment, animalmodels for diseases and disorders involving 53BP2:53BP2-IP complexes and53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 are provided. These include but arenot limited to: cell proliferative disorders including cancer,degenerative disorders involving cellular apoptosis, and benignhypertrophy. Such animals can be initially produced by promotinghomologous recombination or insertional mutagenisis between an 53BP2 anda 53BP2-IP genes or between 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 gene inthe chromosome and exogenous 53BP2 and 53BP2-IP genes or 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 genes that have been rendered biologicallyinactive or deleted (preferably by insertion of a heterologous sequence,e.g., an antibiotic resistance gene). In a preferred aspect, thishomologous recombination is carried out by transforming embryo-derivedstem (ES) cells with a vector containing the insertionally inactivated53BP2 and 53BP2-IP gene or 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 gene, suchthat homologous recombination occurs, followed by injecting the ES cellsinto a blastocyst, and implanting the blastocyst into a foster mother,followed by the birth of the chimeric animal (“knockout animal”) inwhich a 53BP2 and a 53BP2-IP gene or 53B2-IP1, 53BP2-IP2, or 53BP2-IP3gene has been inactivated or deleted (see Capecchi, 1989, Science244:1288-1292). The chimeric animal can be bred to produce additionalknockout animals. Such animals can be mice, hamsters, sheep, pigs,cattle, etc., and are preferably non-human mammals. In a specificembodiment, a knockout mouse is produced.

Such knockout animals are expected to develop or be predisposed todeveloping diseases or disorders involving, but not restricted to, cellproliferative disorders including cancer and benign hypertrophy, variousdisorders involving cellular apoptosis and cellular differentiation,autoimmune diseases, etc., and thus can have use as animal models ofsuch diseases and disorders, e.g., to screen for or test molecules(e.g., potential Therapeutics) for the ability to inhibit cellproliferative, autoimmune, and other diseases.

In a different embodiment of the invention, transgenic animals that haveincorporated and express (or overexpress or misexpress) a functional53BP2 and 53BP2-IP gene or a functional 53BP2-IP1, 53BP2-IP2, 53BP2-IP3gene, e.g. by introducing the 53BP2 and 53BP2-IP genes or the 53BP2-IP1,53BP2-IP2, or 53BP2-IP3 genes under the control of a heterologouspromoter (i.e. a promoter that is not the native 53BP2 or 53BP2-IPpromoter) that either overexpresses the protein or proteins or expressesthem in tissues not normally expressing the complexes or proteins canhave use as animal models of diseases and disorders characterized byelevated levels of 53BP2:53BP2-IP complexes or 53BP2-IP1, 53BP2-IP2, or53BP2-IP3 proteins. Such animals can be used to screen for or testmolecules for the ability to treat or prevent the diseases and disorderscited supra.

In one embodiment, the invention provides a recombinant non-human animalin which both an endogenous 53BP2 gene and an endogenous 53BP2-IP geneselected from the group consisting of β-tubulin, p62, hnRNP G,53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 have been deleted or inactivated byhomologous recombination or insertional mutagenesis of said animal or anancestor thereof. In another embodiment, the invention provides arecombinant non-human animal containing both a 53BP2 gene and a 53BP2-IPgene selected from the group consisting of β-tubulin, p62, hnRNP G,53BP2-IP1, 53BP2-IP2, and 53BP2-IP3, in which the 53BP2 gene is underthe control of a promoter that is not the native 53BP2 gene promoter andthe 53BP2-IP gene is under the control of a promoter that is not thenative 53BP2-IP gene promoter. In a specific embodiment, the inventionprovides a recombinant non-human animal containing a transgenecomprising a nucleic acid sequence encoding a chimeric proteincomprising a fragment of 53BP2 of at least 6 amino acids fused via acovalent bond to a fragment of a 53BP2-IP protein of at least 6 aminoacids.

The invention also provides a recombinant non-human animal in which anendogenous 53BP2-IP gene selected from the group consisting of a53BP2-IP1 gene, a 53BP2-IP2 gene, and a 53BP2-IP3 gene has been deletedor inactivated by homologous recombination or insertional mutagenesis ofsaid animal or an ancestor thereof.

EXAMPLES Identification of 53BP2:53BP2-IP Complexes

A modified, improved yeast two hybrid system was used to identifyprotein interactions. Yeast is a eukaryote, and therefore anyintermolecular protein interactions detected in this type of systemdemonstrate protein interactions that occur under physiologicalconditions (Chien et al., 1991, Proc. Natl. Acad. Sci. USA 88:9578-9581.) Expression vectors were constructed to encode two hybridproteins. For a “forward” screen, one hybrid consisted of the DNAbinding domain of the yeast transcriptional activator Gal4 fused to aportion of 53BP2. The other hybrid consisted of the Gal4 activatordomain fused to “prey” protein sequences encoded by a mammalian cDNAlibrary. In a “reverse” screen, the portion of 53BP2 was fused to theGal4 activator domain, and the prey protein sequences of the mammaliancDNA library were fused to the DNA binding domain, but the assay wasotherwise identically performed. Each of the vectors was then insertedinto complementary (a and alpha) mating types of yeast using methodsknown in the art (Chien et al.,1991, supra). Mating was carried out toexpress both vector constructs within the same yeast cells, thusallowing interaction to occur. Interaction between the bait andprey-domains led to transcriptional activation of reporter genescontaining cis-binding elements for Gal4. The reporter genes encodingthe indicator protein beta-galactosidase, and metabolic markers foruracil and histidine auxotrophy, were included in specific fashion inone or the other of the yeast strains used in the mating. In this way,yeast were selected for successful mating, expression of both fusionconstruct, and expression of 53BP2-IPs. Yeast clones that containedinteracting regions were picked and grown in individual wells ofmicrotiter plates. The plasmids containing the 53BP2-IPs were thenisolated and characterized.

The prey cDNAs were obtained from a fetal brain cDNA library of 1.5×10⁶independent isolates. The library was synthesized from Xho 1-dT₁₅ primedfetal brain mRNA (from five male/female 19-22 week fetuses) that wasdirectionally cloned into pBD-Gal4, a yeast Gal4 DNA binding domaincloning vector including the TRYP gene for selection in yeast deficientin tryptophan biosynthesis.

A reverse screen was used to test the interaction of prey cDNA productsagainst an array of 22 bait proteins, one of which was encoded by the53BP2 nucleotide sequence of nucleotides 2866-3771 as depicted in FIG. 1(SEQ ID NO:l), including amino acids 704-1005 of the 53BP2 amino acidsequence at the C-terminus of 53BP2, as depicted in FIG. 1 (SEQ IDNO:2). The bait fragment was amplified from a Clontech λgt11 library bypolymerase chain reaction (PCR) using the forward primer^(5′)GGACTAGGCCGAGGTGGCCTCTCCAGGCCTTGATTATGAGCCTG^(3′) (SEQ ID NO:14)and the reverse primer^(5′)GGACTAGGCCTCCTCGGCCCTACCTCTGCACTATGTCACTGATTTC^(3′) (SEQ ID NO:15)by standard techniques. The fragment was cloned into the Sfi I site ofthe vector pACT-Sfi I, constructed by introducing an Sfi I-containingpolylinker into the vector pACT2 (Clontech). This vector is a yeastactivation domain cloning vector that contains the LEU2 gene forselection in yeast strains deficient in leucine biosynthesis. The baitwas sequenced to confirm that PCR amplification reproduced an accuratecopy of the 53BP2 sequence (FIG. 1). This test determined that, aspredicted, the bait sequence encoded an interacting domain identical tothe human 53BP2 beginning at amino acid 704 (FIG. 1).

The bait was transformed by lithium acetate/polyethylene glycoltransformation (Ito et al., 1983, J. Bacteriol. 153:163-168) into theyeast strain N106^(r) (mating type a, ura3, his3, ade2, trp1, leu2,gal4, gal80, cyh^(r), Lys2::GAL1_(UAS)-HIS3_(TATA)-HIS3, ura3::GAL1_(UAS)-GAL_(TATA)-lacZ), while the prey sequences weretransformed into the yeast strain YULH (mating type alpha, ura3, his3,lys2, Ade2, trp1, leu2, gal4, gal80, GAL1-lacZ, GAL1-URA3). The twotransformed populations were then mated using standard methods in theart (Sherman et al., eds., 1991, Getting Started with Yeast, Vol. 194,Academic Press, New York). Briefly, cells were grown until mid-to-latelog phase on media that selected for the presence of the appropriateplasmids. The two mating strains, alpha and a, were then, diluted inYAPD media (Sherman et al., eds., 1991, Getting Started with Yeast, Vol.194, Academic Press, New York), filtered onto nitrocellulose membranes,and incubated at 30 degrees Celsius for 6-8 hours. The cells were thentransferred to media selective for the desired diploids, i.e., yeastharboring reporter genes for beta-galactosidase, uracil auxotrophy, andhistidine auxotrophy, and expression of the vectors encoding the baitand prey. The mating products were plated on SC (synthetic complete)(Kaiseret al. eds., 1994, Methods in Genetics, 1994 ed., Cold SpringHarbor Press, New York, p. 209) media lacking adenine and lysine (toselect for successful mating), leucine and tryptophan (to select forexpression of genes encoded by both the bait and prey plasmids), anduracil and histidine (to select for protein interactions). This mediumis herein referred to as SCS medium, for SC Selective medium.

Selected clones were tested for expression of β-galactosidase to confirmthe formation of a 53BP2:53BP2-IP interaction. Filter-liftβ-galactosidase assays were performed as modified from the protocol ofBreeden and Nasmyth (1985, Cold Spring Harbor Quant. Biol. 50:643-650).Colonies were patched onto SCS plates, grown overnight, and replicaplated onto Whatman No. 1 filters. The filters were then assayed forβ-galactosidase activity. Colonies that were positive turned a visibleblue.

Cells in colonies positive for protein interaction contained admixtureof DNA-binding and activation-domain plasmids. These cells wereindividually plated, and regrown as single isolates in individual wellsof 96-well plates. Ten microliters of each isolate was lysed, theinserts within the pACT2 and pASSPiI plasmids were amplified bypolymerase chain reaction using primers specific for the flankingsequences of each vector, and approximately 200 amino-terminal bases ofeach insert was determined using an ABI 377 sequenator. Comparison toknown sequences was made using the “Blast” program publicly availablethrough the National Center for Biotechnology Information. Two of theinserts were identified as β-tubulin, the others identified as p62,hnRNP G, and the insert encoding 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3.Specifically, the inserts contained nucleotides 830-1398 and 895-1398 ofthe coding sequence for β-tubulin as depicted in FIG. 2 (SEQ ID NO:3),nucleotides 929-1435 of the nucleotide sequence of p62 as depicted inFIG. 3 (SEQ ID NO:5), nucleotides 273-1322 of hnRNP G as depicted inFIG. 4 (SEQ ID NO:7), and the sequence depicted in FIG. 5 (encoding inpart 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3). The determined nucleic acidsequences and corresponding amino acid sequences of β-tubulin, p62,hnRNP G, and 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 are shown in FIGS. 2-4,and 9-13, respectively. A summary of the 53BP2 and 53BP2-IP interactingdomains is shown in FIG. 6.

Verification of the Specificity of the 53BP2:Beta-tubulin, P62, hnRNP G,and 53BP2-IP1/2/3 Interactions

To test for the specificity of bait:prey interaction, two general testswere first performed. In the first instance, N106^(r) cells were createdthat express the individual plasmids encoding 53BP2, β-tubulin, p62,hnRNP G, and the sequences encoding 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3.These yeast cells were plated on SCS plates, grown overnight, andexamined for growth. No growth was found for all five proteins,confirming that they were not “self-activating” proteins, that is, theseproteins require interaction with a second protein domain for afunctional activation complex.

In the second instance, plasmids containing β-tubulin, p62, hnRNP G, and53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 inserts were transformed into strainYULH (mating type alpha and mated with yeast strain N106^(r) (matingtype a) expressing proteins other than 53BP2. Promiscuous binders, thatis, inserts able to bind with many other proteins in a non-specificfashion, would interact non-specifically with non-53BP2 domains, andwould be discarded as non-specific interactants. 53BP2 failed tointeract with pRb (GenBank Acc. No. M28419, Lee et al., 1987, Nature329: 642-645), the trk oncogene (GenBank Acc. No. X03541, Martin-Zanca,et al., 1986, Nature 319: 743-748), EST M62042 (Adams et al., 1991,Science 252: 1651-1656), Ral GDS (GenBank Acc. No. U14417, Hofer, etal., 1994, Proc. Natl. Acad. Sci. U.S.A. 91: 11089-11093) or E2F(GenBank Acc. No. X86096, direct submission). β-tubulin, p62, hnRNP G,and the sequences encoding 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 did notinteract with MDM2 (GenBank Acc. No. M92424, Oliner et al., 1994, directsubmission), CAS (GenBank Acc. No. U33286, Brinkmann et al., 1995, Proc.Natl. Acad. Sci. U.S.A. 92: 10427-10431), and PA9 (GenBank Acc. No.S82076, Yang et al., 1996, Carcinogenesis 17:563-567) Specifically, lackof growth for p62 and β-tubulin interactions with MDM2 is depicted inFIG. 7.

To recapitulate the detected interactions, and further demonstrate theirspecificity, the isolated bait plasmid for 53BP2, along with baitplasmids for MDM2 and human bait protein 1 (B1) were used to transformyeast strain N106^(r) (mating type a). The interacting domains from p62and β-tubulin were transformed into strain YULH (mating type alpha). Thetransformants were reamplified, and a mating performed to recapitulatethe identified 53BP2:53BP2-IP interactions. 53BP2 complexed specificallywith β-tubulin and p62, but not with two human proteins, H1 and H2. Asillustrated in FIG. 7, the intersection of the 53BP2 row (bottom) withthe β-tubulin and p62 columns indicates growth (i.e. a positiveinteraction), but the intersection of the 53BP2 row with the columns forH1 and H2 indicates no growth, i.e., no protein interaction. The knowninteraction between 53BP2 and PP1-alpha (Helps et al., 1995, FEBS Letts.377: 295-300) was confirmed, as shown in FIG. 7, (intersection of column3, row 3). As described above, β-tubulin and p62 failed to interact withMDM2 and B1. Mating of PP1-alpha and B1 confirmed an interactionpreviously found in our studies (FIG. 7; Nandabalan et al., 1997,unpublished).

Assembly of the Sequence Encoding 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3

One identified prey sequence was identical to EST R72810 (GenBank database “dbest”). The database contained other EST sequences thatoverlapped the EST R72810 sequence. Thus, it was possible to findcontiguous sequences to extend the nucleotide sequence both 3′ and 5′termini of the EST R72810. The general procedure utilized is illustratedin FIG. 8. The National Center for Biotechnology Information (N.C.B.I.)“Blast” Program was used to compare the EST R72810, and compared to allsequences in the non-redundant nucleotide data bases “NRDB,” acompilation of GenBank+EMBL+DDBJ+PDB sequences (but no EST, STS, GSS, orHTGS sequences), as defined in the NCBI Blast program, “month,” whichincludes all new or revised GenBank+EMBL+DDBJ+PDB sequences released inthe last 30 days, and “dbest,” a non-redundant database ofGenBank+EMBL+DDBJ EST divisions. Sequences that aligned with 95% orgreater identity at the nucleic acid level over their termini of atleast 30 bases were utilized if the alignment resulted in 5′ extensionor 3′ extension of the EST R72810 sequence. Once this first assembly wascomplete, the extended sequence was again subject to the Blastcomparison to detect new homologies to the added extensions. Thesequence was extended in both directions until new related sequencesthat allowed extension of the assembled sequence were no longerdetected.

The extended EST R72810 sequence is illustrated in FIG. 9. The nucleicacid sequence of the original EST R72810 sequence is shown in boldlettering. For 5′ extension, a long overlap was found with EST C17385,the nucleotide sequence of which is denoted by bold underline (Fujiwaraet al., GenBank direct submission, Sep. 9, 1996). For 3′ extension,overlap with EST AA464793 (Hillier et al., 1997, WashU-Merck ESTProject), shown in boxed lettering, was detected. Additional overlapbetween EST AA464793 and EST AA479761 (Hillier et al., 1997, WashU-NCIHuman EST Project), shown in bold, italic lettering, was used forfurther 3′ extension, to complete the EST assembly process. The completeassembled sequence of 915 bases is shown in FIG. 9.

The assembled EST sequence was subjected to a further searches of theNRDB and “month” nucleic acid data bases to detect homologies to knownprotein sequences that were not detected over the shorter span of theoriginal EST sequence. This was necessary where significant homology wasnot detected during the EST assembly process to proteins in the NRDB and“month” data bases, as was the case with the assembled EST R72810. Nosignificant homologies to known proteins were detected for the extendedEST R72810 utilizing this analysis.

Further, the sequence was analyzed by the NCBI program “ORF Finder” thatperformed translations in all three forward reading frames of theassembled DNA sequence (FIGS. 10A-F). EST R72810 (Hillier et al., 1995,GenBank Direct Submission Jun. 2, 1995) was obtained from thedirectionally-cloned Soares breast library 2NbHBst, and thus thedirection of translation of the extended EST is known as 5′ to 3′.Within the three translations, three possible open reading frames(“ORFs”) were identified. Open reading frames greater than 60 aminoacids following an initiator codon or an ORF with no initiatormethionine encoded at the 5′ end were determined to be possible proteinproducts, and were submitted for Blast searching against the proteindata base NRDB, a non-redundant compilation of GenBank CDStranslations+PDB+SwissProt+PIR SwissProt sequences, and “month,” whichincludes all new or revised GenBank CDS translation+PDB+SwissProt+PIRsequences released in the last 30 days.

Three possible protein candidates were identified by the hypotheticaltranslation. The first candidate, encoded in Translation Frame #3 (FIG.10F), encompasses an open reading frame from amino acid 1 (arg) to aminoacid 286 (gln) that precedes an opal translational stop codon. Thistranslational frame has no initiator methionine codon (ATG) and so mustextend further 5′ than the assembled sequence. The second proteincandidate, encoded in Translation Frame #2 (FIG. 10D), extends from theinitiator codon ATG in position 147 (met) to a gln residue, 69 aminoacids downstream, that precedes an amber translational stop codon. Thethird protein candidate, encoded in Translation Frame #1 (FIG. 10B),extends from a glycine residue at position 1 (no initiation methioninecodon was found in this reading frame) to a glycine at position 33, thatprecedes a TAA stop codon. None of these proteins displayed significanthomology to any known protein at the nucleic acid or amino acid levels.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

15 1 4534 DNA Homo sapiens CDS (757)..(3771) 1 gtcacgagcg tcgaagagacaaagccgcgt cagggggccc ggccggggcg ggggagcccg 60 gggcttgttg gtgccccagcccgcgcggag ggcccttcgg acccgcgcgc cgccgctgcc 120 gccgccgccg cctcgcaacaggtccgggcg gcctcgctct ccgctcccct cccccgcatc 180 cgcgaccctc cggggcacctcagctcggcc ggggccgcag tctggccacc cgcttccatg 240 cggttcgggt ccaagatgatgccgatgttt cttaccgtgt atctcagtaa caatgagcag 300 cacttcacag aagttccagttactccagaa acaatatgca gagacgtggt ggatctgtgc 360 aaagaacccg gcgagagtgattgccatttg gctgaagtgt ggtgtggctc tgtagagata 420 gagtttcatc atgttggccaggatggtctc gatctcctga ccttgtgatc cgcctgcctc 480 ggcctcccaa agtgctggattacaggtgtg agccaccacg atcagcctct agtgtttaaa 540 aaagaacgtc cagttgcggataatgagcga atgtttgatg ttcttcaacg atttggaagt 600 cagaggaacg aagttcgcttcttccttcgt catgaacgcc cccctggcag ggacattgtg 660 agtggaccaa gatctcaggatccaagttta aaaagaaatg gtgtaaaagt tcctggtgaa 720 tatcgaagaa aggagaacggtgttaatagt cctagg atg gat ctg act ctt gct 774 Met Asp Leu Thr Leu Ala 15 gaa ctt cag gaa atg gca tct cgc cag cag caa cag att gaa gcc cag 822Glu Leu Gln Glu Met Ala Ser Arg Gln Gln Gln Gln Ile Glu Ala Gln 10 15 20caa caa ttg ctg gca act aag gaa cag cgc tta aag ttt ttg aaa caa 870 GlnGln Leu Leu Ala Thr Lys Glu Gln Arg Leu Lys Phe Leu Lys Gln 25 30 35 caagat cag cga caa cag caa caa gtt gct gag cag gag aaa ctt aaa 918 Gln AspGln Arg Gln Gln Gln Gln Val Ala Glu Gln Glu Lys Leu Lys 40 45 50 agg ctaaaa gaa ata gct gag aat cag gaa gct aag cta aaa aaa gtg 966 Arg Leu LysGlu Ile Ala Glu Asn Gln Glu Ala Lys Leu Lys Lys Val 55 60 65 70 aga gcactt aaa ggc cac gtg gaa cag aag aga cta agc aat ggg aaa 1014 Arg Ala LeuLys Gly His Val Glu Gln Lys Arg Leu Ser Asn Gly Lys 75 80 85 ctt gtg gaggaa att gaa cag atg aat aat ttg ttc cag caa aaa cag 1062 Leu Val Glu GluIle Glu Gln Met Asn Asn Leu Phe Gln Gln Lys Gln 90 95 100 agg gag ctcgtc ctg gct gtg tca aaa gta gaa gaa ctg acc agg cag 1110 Arg Glu Leu ValLeu Ala Val Ser Lys Val Glu Glu Leu Thr Arg Gln 105 110 115 cta gag atgctc aag aac ggc agg atc gac agc cac cat gac aat cag 1158 Leu Glu Met LeuLys Asn Gly Arg Ile Asp Ser His His Asp Asn Gln 120 125 130 tct gca gtggct gag ctt gat cgc ctc tat aag gag ctg cag cta aga 1206 Ser Ala Val AlaGlu Leu Asp Arg Leu Tyr Lys Glu Leu Gln Leu Arg 135 140 145 150 aac aaattg aat caa gag cag aat gcc aag cta caa caa cag agg gag 1254 Asn Lys LeuAsn Gln Glu Gln Asn Ala Lys Leu Gln Gln Gln Arg Glu 155 160 165 tgt ttgaat aag cgt aat tca gaa gtg gca gtc atg gat aag cgt gtt 1302 Cys Leu AsnLys Arg Asn Ser Glu Val Ala Val Met Asp Lys Arg Val 170 175 180 aat gagctg agg gac cgg ctg tgg aag aag aag gca gct cta cag caa 1350 Asn Glu LeuArg Asp Arg Leu Trp Lys Lys Lys Ala Ala Leu Gln Gln 185 190 195 aaa gaaaat cta cca gtt tca tct gat gga aat ctt ccc cag caa gcc 1398 Lys Glu AsnLeu Pro Val Ser Ser Asp Gly Asn Leu Pro Gln Gln Ala 200 205 210 gcg tcagcc cca agc cgt gtg gct gca gta ggt ccc tat atc cag tca 1446 Ala Ser AlaPro Ser Arg Val Ala Ala Val Gly Pro Tyr Ile Gln Ser 215 220 225 230 tctact atg cct cgg atg ccc tca agg cct gaa ttg ctg gtg aag cca 1494 Ser ThrMet Pro Arg Met Pro Ser Arg Pro Glu Leu Leu Val Lys Pro 235 240 245 gccctg ccg gat ggt tcc ttg gtc att cag gct tca gag ggg ccg atg 1542 Ala LeuPro Asp Gly Ser Leu Val Ile Gln Ala Ser Glu Gly Pro Met 250 255 260 aaaata cag aca ctg ccc aac atg aga tct ggg gct gct tca caa act 1590 Lys IleGln Thr Leu Pro Asn Met Arg Ser Gly Ala Ala Ser Gln Thr 265 270 275 aaaggc tct aaa atc cat cca gtt ggc cct gat tgg agt cct tca aat 1638 Lys GlySer Lys Ile His Pro Val Gly Pro Asp Trp Ser Pro Ser Asn 280 285 290 gcagat ctt ttc cca agc caa ggc tct gct tct gta cct caa agc act 1686 Ala AspLeu Phe Pro Ser Gln Gly Ser Ala Ser Val Pro Gln Ser Thr 295 300 305 310ggg aat gct ctg gat caa gtt gat gat gga gag gtt ccg ctg agg gag 1734 GlyAsn Ala Leu Asp Gln Val Asp Asp Gly Glu Val Pro Leu Arg Glu 315 320 325aaa gag aag aaa gtg cgt ccg ttc tca atg ttt gat gca gta gac cag 1782 LysGlu Lys Lys Val Arg Pro Phe Ser Met Phe Asp Ala Val Asp Gln 330 335 340tcc aat gcc cca cct tcc ttt ggt act ctg agg aag aac cag agc agt 1830 SerAsn Ala Pro Pro Ser Phe Gly Thr Leu Arg Lys Asn Gln Ser Ser 345 350 355gaa gat atc ttg cgg gat gct cag gtt gca aat aaa aat gtg gct aaa 1878 GluAsp Ile Leu Arg Asp Ala Gln Val Ala Asn Lys Asn Val Ala Lys 360 365 370gta cca cct cct gtt cct aca aaa cca aaa cag att aat ttg cct tat 1926 ValPro Pro Pro Val Pro Thr Lys Pro Lys Gln Ile Asn Leu Pro Tyr 375 380 385390 ttt gga caa act aat cag cca cct tca gac att aag cca gac gga agt 1974Phe Gly Gln Thr Asn Gln Pro Pro Ser Asp Ile Lys Pro Asp Gly Ser 395 400405 tct cag cag ttg tca aca gtt gtt ccg tcc atg gga act aaa cca aaa 2022Ser Gln Gln Leu Ser Thr Val Val Pro Ser Met Gly Thr Lys Pro Lys 410 415420 cca gca ggg cag cag ccg aga gtg ctg cta tct ccc agc ata cct tcg 2070Pro Ala Gly Gln Gln Pro Arg Val Leu Leu Ser Pro Ser Ile Pro Ser 425 430435 gtt ggc caa gac cag acc ctt tct cca ggt tct aag caa gaa agt cca 2118Val Gly Gln Asp Gln Thr Leu Ser Pro Gly Ser Lys Gln Glu Ser Pro 440 445450 cct gct gct gcc gtc cgg ccc ttt act ccc cag cct tcc aaa gac acc 2166Pro Ala Ala Ala Val Arg Pro Phe Thr Pro Gln Pro Ser Lys Asp Thr 455 460465 470 tta ctt cca ccc ttc aga aaa ccc cag acc gtg gca gca agt tca ata2214 Leu Leu Pro Pro Phe Arg Lys Pro Gln Thr Val Ala Ala Ser Ser Ile 475480 485 tat tcc atg tat acg caa cag cag gcg cca gga aaa aac ttc cag cag2262 Tyr Ser Met Tyr Thr Gln Gln Gln Ala Pro Gly Lys Asn Phe Gln Gln 490495 500 gct gtg cag agc gcg ttg acc aag act cat acc aga ggg cca cac ttt2310 Ala Val Gln Ser Ala Leu Thr Lys Thr His Thr Arg Gly Pro His Phe 505510 515 tca agt gta tat ggt aag cct gta att gct gct gcc cag aat caa cag2358 Ser Ser Val Tyr Gly Lys Pro Val Ile Ala Ala Ala Gln Asn Gln Gln 520525 530 cag cac cca gag aac att tat tcc aat agc cag ggc aag cct ggc agt2406 Gln His Pro Glu Asn Ile Tyr Ser Asn Ser Gln Gly Lys Pro Gly Ser 535540 545 550 cca gaa cct gaa aca gag cct gtt tct tca gtt cag gag aac catgaa 2454 Pro Glu Pro Glu Thr Glu Pro Val Ser Ser Val Gln Glu Asn His Glu555 560 565 aac gaa aga att cct cgg cca ctc agc cca act aaa tta ctg cctttc 2502 Asn Glu Arg Ile Pro Arg Pro Leu Ser Pro Thr Lys Leu Leu Pro Phe570 575 580 tta tct aat cct tac cga aac cag agt gat gct gac cta gaa gcctta 2550 Leu Ser Asn Pro Tyr Arg Asn Gln Ser Asp Ala Asp Leu Glu Ala Leu585 590 595 cga aag aaa ctg tct aac gca cca agg cct cta aag aaa cgt agttct 2598 Arg Lys Lys Leu Ser Asn Ala Pro Arg Pro Leu Lys Lys Arg Ser Ser600 605 610 att aca gag cca gag ggt cct aat ggg cca aat att cag aag ctttta 2646 Ile Thr Glu Pro Glu Gly Pro Asn Gly Pro Asn Ile Gln Lys Leu Leu615 620 625 630 tat cag agg acc acc ata gcg gcc atg gag acc atc tct gtccca tca 2694 Tyr Gln Arg Thr Thr Ile Ala Ala Met Glu Thr Ile Ser Val ProSer 635 640 645 tac cca tcc aag tca gct tct gtg act gcc agc tca gaa agccca gta 2742 Tyr Pro Ser Lys Ser Ala Ser Val Thr Ala Ser Ser Glu Ser ProVal 650 655 660 gaa atc cag aat cca tat tta cat gtg gag ccc gaa aag gaggtg gtc 2790 Glu Ile Gln Asn Pro Tyr Leu His Val Glu Pro Glu Lys Glu ValVal 665 670 675 tct ctg gtt cct gaa tca ttg tcc cca gag gat gtg ggg aatgcc agt 2838 Ser Leu Val Pro Glu Ser Leu Ser Pro Glu Asp Val Gly Asn AlaSer 680 685 690 aca gag aac agt gac atg cca gct cct tct cca ggc ctt gattat gag 2886 Thr Glu Asn Ser Asp Met Pro Ala Pro Ser Pro Gly Leu Asp TyrGlu 695 700 705 710 cct gag gga gtc cca gac aac agc cca aat ctc cag aataac cca gaa 2934 Pro Glu Gly Val Pro Asp Asn Ser Pro Asn Leu Gln Asn AsnPro Glu 715 720 725 gaa cca aat cca gag gct cca cat gtg ctt gat gtg tacctg gag gag 2982 Glu Pro Asn Pro Glu Ala Pro His Val Leu Asp Val Tyr LeuGlu Glu 730 735 740 tac cct cca tac cca ccc cca cca tac cca tct ggg gagcct gaa ggg 3030 Tyr Pro Pro Tyr Pro Pro Pro Pro Tyr Pro Ser Gly Glu ProGlu Gly 745 750 755 ccc gga gaa gac tcg gtg agc atg cgc ccg cct gaa atcacc ggg cag 3078 Pro Gly Glu Asp Ser Val Ser Met Arg Pro Pro Glu Ile ThrGly Gln 760 765 770 gtc tct ctg cct cct ggt aaa agg aca aac ttg cgt aaaact ggc tca 3126 Val Ser Leu Pro Pro Gly Lys Arg Thr Asn Leu Arg Lys ThrGly Ser 775 780 785 790 gag cgt atc gct cat gga atg agg gtg aaa ttc aacccc ctt gct tta 3174 Glu Arg Ile Ala His Gly Met Arg Val Lys Phe Asn ProLeu Ala Leu 795 800 805 ctg cta gat tcg tct ttg gag gga gaa ttt gac cttgta cag aga att 3222 Leu Leu Asp Ser Ser Leu Glu Gly Glu Phe Asp Leu ValGln Arg Ile 810 815 820 att tat gag gtt gat gac cca agc ctg ccc aat gatgaa ggc atc acg 3270 Ile Tyr Glu Val Asp Asp Pro Ser Leu Pro Asn Asp GluGly Ile Thr 825 830 835 gct ctt cac aat gct gtg tgt gca ggc cac aca gaaatc gtt aag ttc 3318 Ala Leu His Asn Ala Val Cys Ala Gly His Thr Glu IleVal Lys Phe 840 845 850 ctg gta cag ttt ggt gta aat gta aat gct gct gatagt gat gga tgg 3366 Leu Val Gln Phe Gly Val Asn Val Asn Ala Ala Asp SerAsp Gly Trp 855 860 865 870 act cca tta cat tgt gct gcc tca tgt aac aacgtc caa gtg tgt aag 3414 Thr Pro Leu His Cys Ala Ala Ser Cys Asn Asn ValGln Val Cys Lys 875 880 885 ttt ttg gtg gag tca gga gcc gct gtg ttt gccatg acc tac agt gac 3462 Phe Leu Val Glu Ser Gly Ala Ala Val Phe Ala MetThr Tyr Ser Asp 890 895 900 atg cag act gct gca gat aag tgc gag gaa atggag gaa ggc tac act 3510 Met Gln Thr Ala Ala Asp Lys Cys Glu Glu Met GluGlu Gly Tyr Thr 905 910 915 cag tgc tcc caa ttt ctt tat gga gtt cag gagaag atg ggc ata atg 3558 Gln Cys Ser Gln Phe Leu Tyr Gly Val Gln Glu LysMet Gly Ile Met 920 925 930 aat aaa gga gtc att tat gcg ctt tgg gat tatgaa cct cag aat gat 3606 Asn Lys Gly Val Ile Tyr Ala Leu Trp Asp Tyr GluPro Gln Asn Asp 935 940 945 950 gat gag ctg ccc atg aaa gaa gga gac tgcatg aca atc atc cac agg 3654 Asp Glu Leu Pro Met Lys Glu Gly Asp Cys MetThr Ile Ile His Arg 955 960 965 gaa gac gaa gat gaa atc gaa tgg tgg tgggcg cgc ctt aat gat aag 3702 Glu Asp Glu Asp Glu Ile Glu Trp Trp Trp AlaArg Leu Asn Asp Lys 970 975 980 gag gga tat gtt cca cgt aac ttg ctg ggactg tac cca aga att aaa 3750 Glu Gly Tyr Val Pro Arg Asn Leu Leu Gly LeuTyr Pro Arg Ile Lys 985 990 995 cca aga caa agg agc ttg gcc tgaaacttccacacagaatt ttagtcaatg 3801 Pro Arg Gln Arg Ser Leu Ala 1000 1005aagaattaat ctctgttaag aagaagtaat acgattattt ttggcaaaaa tttcacaaga 3861cttattttaa tgacaatgta gcttgaaagc gatgaagaat gtctctagaa gagaatgaag 3921gattgaagaa ttcaccatta gaggacattt agcgtgatga aataaagcat ctacgtcagc 3981aggccatact gtgttggggc aaaggtgtcc cgtgtagcac tcagataagt atacagcgac 4041aatcctgttt tctacaagaa tcctgtctag taaataggat catttattgg gcagttggga 4101aatcagctct ctgtcctgtt gagtgttttc agcagctgct cctaaaccag tcctcctgcc 4161agaaaggacc agtgccgtca catcgctgtc tctgattgtc cccggcacca gcaggccttg 4221gggctcactg aaggctcgaa ggcactgcac accttgtata ttgtcagtga agaacgttag 4281ttggttgtca gtgaacaata actttattat atgagttttt gtagcatctt aagaattata 4341catatgtttg aaatattgaa actaagctac agtaccagta attagatgta gaatcttgtt 4401tgtaggctga attttaatct gtatttattg tcttttgtat ctcagaaatt agaaacttgc 4461tacagactta cccgtaatat ttgtcaagat catagctgac tttaaaaaca gttgtaataa 4521actttttgat gct 4534 2 1005 PRT Homo sapiens 2 Met Asp Leu Thr Leu AlaGlu Leu Gln Glu Met Ala Ser Arg Gln Gln 1 5 10 15 Gln Gln Ile Glu AlaGln Gln Gln Leu Leu Ala Thr Lys Glu Gln Arg 20 25 30 Leu Lys Phe Leu LysGln Gln Asp Gln Arg Gln Gln Gln Gln Val Ala 35 40 45 Glu Gln Glu Lys LeuLys Arg Leu Lys Glu Ile Ala Glu Asn Gln Glu 50 55 60 Ala Lys Leu Lys LysVal Arg Ala Leu Lys Gly His Val Glu Gln Lys 65 70 75 80 Arg Leu Ser AsnGly Lys Leu Val Glu Glu Ile Glu Gln Met Asn Asn 85 90 95 Leu Phe Gln GlnLys Gln Arg Glu Leu Val Leu Ala Val Ser Lys Val 100 105 110 Glu Glu LeuThr Arg Gln Leu Glu Met Leu Lys Asn Gly Arg Ile Asp 115 120 125 Ser HisHis Asp Asn Gln Ser Ala Val Ala Glu Leu Asp Arg Leu Tyr 130 135 140 LysGlu Leu Gln Leu Arg Asn Lys Leu Asn Gln Glu Gln Asn Ala Lys 145 150 155160 Leu Gln Gln Gln Arg Glu Cys Leu Asn Lys Arg Asn Ser Glu Val Ala 165170 175 Val Met Asp Lys Arg Val Asn Glu Leu Arg Asp Arg Leu Trp Lys Lys180 185 190 Lys Ala Ala Leu Gln Gln Lys Glu Asn Leu Pro Val Ser Ser AspGly 195 200 205 Asn Leu Pro Gln Gln Ala Ala Ser Ala Pro Ser Arg Val AlaAla Val 210 215 220 Gly Pro Tyr Ile Gln Ser Ser Thr Met Pro Arg Met ProSer Arg Pro 225 230 235 240 Glu Leu Leu Val Lys Pro Ala Leu Pro Asp GlySer Leu Val Ile Gln 245 250 255 Ala Ser Glu Gly Pro Met Lys Ile Gln ThrLeu Pro Asn Met Arg Ser 260 265 270 Gly Ala Ala Ser Gln Thr Lys Gly SerLys Ile His Pro Val Gly Pro 275 280 285 Asp Trp Ser Pro Ser Asn Ala AspLeu Phe Pro Ser Gln Gly Ser Ala 290 295 300 Ser Val Pro Gln Ser Thr GlyAsn Ala Leu Asp Gln Val Asp Asp Gly 305 310 315 320 Glu Val Pro Leu ArgGlu Lys Glu Lys Lys Val Arg Pro Phe Ser Met 325 330 335 Phe Asp Ala ValAsp Gln Ser Asn Ala Pro Pro Ser Phe Gly Thr Leu 340 345 350 Arg Lys AsnGln Ser Ser Glu Asp Ile Leu Arg Asp Ala Gln Val Ala 355 360 365 Asn LysAsn Val Ala Lys Val Pro Pro Pro Val Pro Thr Lys Pro Lys 370 375 380 GlnIle Asn Leu Pro Tyr Phe Gly Gln Thr Asn Gln Pro Pro Ser Asp 385 390 395400 Ile Lys Pro Asp Gly Ser Ser Gln Gln Leu Ser Thr Val Val Pro Ser 405410 415 Met Gly Thr Lys Pro Lys Pro Ala Gly Gln Gln Pro Arg Val Leu Leu420 425 430 Ser Pro Ser Ile Pro Ser Val Gly Gln Asp Gln Thr Leu Ser ProGly 435 440 445 Ser Lys Gln Glu Ser Pro Pro Ala Ala Ala Val Arg Pro PheThr Pro 450 455 460 Gln Pro Ser Lys Asp Thr Leu Leu Pro Pro Phe Arg LysPro Gln Thr 465 470 475 480 Val Ala Ala Ser Ser Ile Tyr Ser Met Tyr ThrGln Gln Gln Ala Pro 485 490 495 Gly Lys Asn Phe Gln Gln Ala Val Gln SerAla Leu Thr Lys Thr His 500 505 510 Thr Arg Gly Pro His Phe Ser Ser ValTyr Gly Lys Pro Val Ile Ala 515 520 525 Ala Ala Gln Asn Gln Gln Gln HisPro Glu Asn Ile Tyr Ser Asn Ser 530 535 540 Gln Gly Lys Pro Gly Ser ProGlu Pro Glu Thr Glu Pro Val Ser Ser 545 550 555 560 Val Gln Glu Asn HisGlu Asn Glu Arg Ile Pro Arg Pro Leu Ser Pro 565 570 575 Thr Lys Leu LeuPro Phe Leu Ser Asn Pro Tyr Arg Asn Gln Ser Asp 580 585 590 Ala Asp LeuGlu Ala Leu Arg Lys Lys Leu Ser Asn Ala Pro Arg Pro 595 600 605 Leu LysLys Arg Ser Ser Ile Thr Glu Pro Glu Gly Pro Asn Gly Pro 610 615 620 AsnIle Gln Lys Leu Leu Tyr Gln Arg Thr Thr Ile Ala Ala Met Glu 625 630 635640 Thr Ile Ser Val Pro Ser Tyr Pro Ser Lys Ser Ala Ser Val Thr Ala 645650 655 Ser Ser Glu Ser Pro Val Glu Ile Gln Asn Pro Tyr Leu His Val Glu660 665 670 Pro Glu Lys Glu Val Val Ser Leu Val Pro Glu Ser Leu Ser ProGlu 675 680 685 Asp Val Gly Asn Ala Ser Thr Glu Asn Ser Asp Met Pro AlaPro Ser 690 695 700 Pro Gly Leu Asp Tyr Glu Pro Glu Gly Val Pro Asp AsnSer Pro Asn 705 710 715 720 Leu Gln Asn Asn Pro Glu Glu Pro Asn Pro GluAla Pro His Val Leu 725 730 735 Asp Val Tyr Leu Glu Glu Tyr Pro Pro TyrPro Pro Pro Pro Tyr Pro 740 745 750 Ser Gly Glu Pro Glu Gly Pro Gly GluAsp Ser Val Ser Met Arg Pro 755 760 765 Pro Glu Ile Thr Gly Gln Val SerLeu Pro Pro Gly Lys Arg Thr Asn 770 775 780 Leu Arg Lys Thr Gly Ser GluArg Ile Ala His Gly Met Arg Val Lys 785 790 795 800 Phe Asn Pro Leu AlaLeu Leu Leu Asp Ser Ser Leu Glu Gly Glu Phe 805 810 815 Asp Leu Val GlnArg Ile Ile Tyr Glu Val Asp Asp Pro Ser Leu Pro 820 825 830 Asn Asp GluGly Ile Thr Ala Leu His Asn Ala Val Cys Ala Gly His 835 840 845 Thr GluIle Val Lys Phe Leu Val Gln Phe Gly Val Asn Val Asn Ala 850 855 860 AlaAsp Ser Asp Gly Trp Thr Pro Leu His Cys Ala Ala Ser Cys Asn 865 870 875880 Asn Val Gln Val Cys Lys Phe Leu Val Glu Ser Gly Ala Ala Val Phe 885890 895 Ala Met Thr Tyr Ser Asp Met Gln Thr Ala Ala Asp Lys Cys Glu Glu900 905 910 Met Glu Glu Gly Tyr Thr Gln Cys Ser Gln Phe Leu Tyr Gly ValGln 915 920 925 Glu Lys Met Gly Ile Met Asn Lys Gly Val Ile Tyr Ala LeuTrp Asp 930 935 940 Tyr Glu Pro Gln Asn Asp Asp Glu Leu Pro Met Lys GluGly Asp Cys 945 950 955 960 Met Thr Ile Ile His Arg Glu Asp Glu Asp GluIle Glu Trp Trp Trp 965 970 975 Ala Arg Leu Asn Asp Lys Glu Gly Tyr ValPro Arg Asn Leu Leu Gly 980 985 990 Leu Tyr Pro Arg Ile Lys Pro Arg GlnArg Ser Leu Ala 995 1000 1005 3 1594 DNA Homo sapiens CDS (64)..(1398) 3gcccgccggt ccacgccgcg caccgctccg agggccagcg ccacccgctc cgcagccggc 60 accatg cgc gag atc gtg cac atc cag gcg ggc cag tgc ggc aac cag 108 Met ArgGlu Ile Val His Ile Gln Ala Gly Gln Cys Gly Asn Gln 1 5 10 15 atc ggcgcc aag ttt tgg gag gtc atc agc gat gag cat ggg atc gac 156 Ile Gly AlaLys Phe Trp Glu Val Ile Ser Asp Glu His Gly Ile Asp 20 25 30 ccc aca ggcagt tac cat gga gac agt gac ttg cag ctg gag aga atc 204 Pro Thr Gly SerTyr His Gly Asp Ser Asp Leu Gln Leu Glu Arg Ile 35 40 45 aac gtg tac tacaat gag gct gct ggt aac aaa tat gta cct cgg gcc 252 Asn Val Tyr Tyr AsnGlu Ala Ala Gly Asn Lys Tyr Val Pro Arg Ala 50 55 60 atc ctg gtg gat ctggag cct ggc acc atg gac tct gtc agg tct gga 300 Ile Leu Val Asp Leu GluPro Gly Thr Met Asp Ser Val Arg Ser Gly 65 70 75 ccc ttc ggc cag atc ttcaga cca gac aac ttc gtg ttc ggc cag agt 348 Pro Phe Gly Gln Ile Phe ArgPro Asp Asn Phe Val Phe Gly Gln Ser 80 85 90 95 gga gcc ggg aat aac tgggcc aag ggc cac tac aca gag gga gcc gag 396 Gly Ala Gly Asn Asn Trp AlaLys Gly His Tyr Thr Glu Gly Ala Glu 100 105 110 ctg gtc gac tcg gtc ctggat gtg gtg agg aag gag tca gag agc tgt 444 Leu Val Asp Ser Val Leu AspVal Val Arg Lys Glu Ser Glu Ser Cys 115 120 125 gac tgt ctc cag ggc ttccag ctg acc cac tct ctg ggg ggc ggc acg 492 Asp Cys Leu Gln Gly Phe GlnLeu Thr His Ser Leu Gly Gly Gly Thr 130 135 140 ggg tcc ggg atg ggc accctg ctc atc agc aag atc cgg gaa gag tac 540 Gly Ser Gly Met Gly Thr LeuLeu Ile Ser Lys Ile Arg Glu Glu Tyr 145 150 155 cca gac cgc atc atg aacacc ttc agc gtc atg ccc tca ccc aag gtg 588 Pro Asp Arg Ile Met Asn ThrPhe Ser Val Met Pro Ser Pro Lys Val 160 165 170 175 tca gac acg gtg gtggag ccc tac aac gcc acc ctc tcg gtc cac cag 636 Ser Asp Thr Val Val GluPro Tyr Asn Ala Thr Leu Ser Val His Gln 180 185 190 ctg gtg gaa aac acagat gaa acc tac tcc att gat aac gag gcc ctg 684 Leu Val Glu Asn Thr AspGlu Thr Tyr Ser Ile Asp Asn Glu Ala Leu 195 200 205 tat gac atc tgc ttccgc acc ctg aag ctg acc acc ccc acc tac ggg 732 Tyr Asp Ile Cys Phe ArgThr Leu Lys Leu Thr Thr Pro Thr Tyr Gly 210 215 220 gac ctc aac cac ctggtg tcg gcc acc atg agc ggg gtc acc acc tgc 780 Asp Leu Asn His Leu ValSer Ala Thr Met Ser Gly Val Thr Thr Cys 225 230 235 ctg cgc ttc ccg ggccag ctg aac gca gac ctg cgc aag ctg gcg gtg 828 Leu Arg Phe Pro Gly GlnLeu Asn Ala Asp Leu Arg Lys Leu Ala Val 240 245 250 255 aac atg gtg cccttc cct cgc ctg cac ttc ttc atg ccc ggc ttc gcg 876 Asn Met Val Pro PhePro Arg Leu His Phe Phe Met Pro Gly Phe Ala 260 265 270 ccc ctg acc agccgg ggc agc cag cag tac cgg gcg ctc acg gtg ccc 924 Pro Leu Thr Ser ArgGly Ser Gln Gln Tyr Arg Ala Leu Thr Val Pro 275 280 285 gag ctc acc cagcag atg ttc gac tcc aag aac atg atg gcc gcc tgc 972 Glu Leu Thr Gln GlnMet Phe Asp Ser Lys Asn Met Met Ala Ala Cys 290 295 300 gac ccg cgc cacggc cgc tac ctg acg gtg gct gcc atc ttc cgg ggc 1020 Asp Pro Arg His GlyArg Tyr Leu Thr Val Ala Ala Ile Phe Arg Gly 305 310 315 cgc atg tcc atgaag gag gtg gac gag cag atg ctc aac gtg cag aac 1068 Arg Met Ser Met LysGlu Val Asp Glu Gln Met Leu Asn Val Gln Asn 320 325 330 335 aag aac agcagc tac ttc gtg gag tgg atc ccc aac aac gtg aag acg 1116 Lys Asn Ser SerTyr Phe Val Glu Trp Ile Pro Asn Asn Val Lys Thr 340 345 350 gcc gtg tgcgac atc ccg ccc cgc ggc ctg aag atg tcg gcc acc ttc 1164 Ala Val Cys AspIle Pro Pro Arg Gly Leu Lys Met Ser Ala Thr Phe 355 360 365 atc ggc aacagc acg gcc atc cag gag ctg ttc aag cgc atc tcc gag 1212 Ile Gly Asn SerThr Ala Ile Gln Glu Leu Phe Lys Arg Ile Ser Glu 370 375 380 cag ttc acggcc atg ttc cgg cgc aag gcc ttc ctg cac tgg tac acg 1260 Gln Phe Thr AlaMet Phe Arg Arg Lys Ala Phe Leu His Trp Tyr Thr 385 390 395 ggc gag ggcatg gac gag atg gag ttc acc gag gcc gag agc aac atg 1308 Gly Glu Gly MetAsp Glu Met Glu Phe Thr Glu Ala Glu Ser Asn Met 400 405 410 415 aac gacctg gtg tcc gag tac cag cag tac cag gac gcc acg gcc gac 1356 Asn Asp LeuVal Ser Glu Tyr Gln Gln Tyr Gln Asp Ala Thr Ala Asp 420 425 430 gaa caaggg gag ttc gag gag gag gag ggc gag gac gag gct 1398 Glu Gln Gly Glu PheGlu Glu Glu Glu Gly Glu Asp Glu Ala 435 440 445 taaaaacttc tcagatcaatcgtgcatcct tagtgaactt ctgttgtcct caagcatggt 1458 ctttctactt gtaaactatggtgctcagtt ttgcctctgt tagaaattca cactgttgat 1518 gtaatgatgt ggaactcctctaaaaattac agtattgtct gtgaaggtat ctatactaat 1578 aaaaaagcat gtgtag 15944 445 PRT Homo sapiens 4 Met Arg Glu Ile Val His Ile Gln Ala Gly Gln CysGly Asn Gln Ile 1 5 10 15 Gly Ala Lys Phe Trp Glu Val Ile Ser Asp GluHis Gly Ile Asp Pro 20 25 30 Thr Gly Ser Tyr His Gly Asp Ser Asp Leu GlnLeu Glu Arg Ile Asn 35 40 45 Val Tyr Tyr Asn Glu Ala Ala Gly Asn Lys TyrVal Pro Arg Ala Ile 50 55 60 Leu Val Asp Leu Glu Pro Gly Thr Met Asp SerVal Arg Ser Gly Pro 65 70 75 80 Phe Gly Gln Ile Phe Arg Pro Asp Asn PheVal Phe Gly Gln Ser Gly 85 90 95 Ala Gly Asn Asn Trp Ala Lys Gly His TyrThr Glu Gly Ala Glu Leu 100 105 110 Val Asp Ser Val Leu Asp Val Val ArgLys Glu Ser Glu Ser Cys Asp 115 120 125 Cys Leu Gln Gly Phe Gln Leu ThrHis Ser Leu Gly Gly Gly Thr Gly 130 135 140 Ser Gly Met Gly Thr Leu LeuIle Ser Lys Ile Arg Glu Glu Tyr Pro 145 150 155 160 Asp Arg Ile Met AsnThr Phe Ser Val Met Pro Ser Pro Lys Val Ser 165 170 175 Asp Thr Val ValGlu Pro Tyr Asn Ala Thr Leu Ser Val His Gln Leu 180 185 190 Val Glu AsnThr Asp Glu Thr Tyr Ser Ile Asp Asn Glu Ala Leu Tyr 195 200 205 Asp IleCys Phe Arg Thr Leu Lys Leu Thr Thr Pro Thr Tyr Gly Asp 210 215 220 LeuAsn His Leu Val Ser Ala Thr Met Ser Gly Val Thr Thr Cys Leu 225 230 235240 Arg Phe Pro Gly Gln Leu Asn Ala Asp Leu Arg Lys Leu Ala Val Asn 245250 255 Met Val Pro Phe Pro Arg Leu His Phe Phe Met Pro Gly Phe Ala Pro260 265 270 Leu Thr Ser Arg Gly Ser Gln Gln Tyr Arg Ala Leu Thr Val ProGlu 275 280 285 Leu Thr Gln Gln Met Phe Asp Ser Lys Asn Met Met Ala AlaCys Asp 290 295 300 Pro Arg His Gly Arg Tyr Leu Thr Val Ala Ala Ile PheArg Gly Arg 305 310 315 320 Met Ser Met Lys Glu Val Asp Glu Gln Met LeuAsn Val Gln Asn Lys 325 330 335 Asn Ser Ser Tyr Phe Val Glu Trp Ile ProAsn Asn Val Lys Thr Ala 340 345 350 Val Cys Asp Ile Pro Pro Arg Gly LeuLys Met Ser Ala Thr Phe Ile 355 360 365 Gly Asn Ser Thr Ala Ile Gln GluLeu Phe Lys Arg Ile Ser Glu Gln 370 375 380 Phe Thr Ala Met Phe Arg ArgLys Ala Phe Leu His Trp Tyr Thr Gly 385 390 395 400 Glu Gly Met Asp GluMet Glu Phe Thr Glu Ala Glu Ser Asn Met Asn 405 410 415 Asp Leu Val SerGlu Tyr Gln Gln Tyr Gln Asp Ala Thr Ala Asp Glu 420 425 430 Gln Gly GluPhe Glu Glu Glu Glu Gly Glu Asp Glu Ala 435 440 445 5 2685 DNA Homosapiens CDS (107)..(1435) 5 ggcttcggtc gctaccgctc ccgctctgcc acccccgccaaccgccgctc gggcctccgt 60 cgctgccgcg tcgctttctc gctccttgga tcgcacatcctcccag atg cag cgc 115 Met Gln Arg 1 cgg gac gac ccc gcc gcg cgc atg agccgg tct tcg ggc cgt agc ggc 163 Arg Asp Asp Pro Ala Ala Arg Met Ser ArgSer Ser Gly Arg Ser Gly 5 10 15 tcc atg gac ccc tcc ggt gcc cac ccc tcggtg cgt cag acg ccg tct 211 Ser Met Asp Pro Ser Gly Ala His Pro Ser ValArg Gln Thr Pro Ser 20 25 30 35 cgg cag ccg ccg ctg cct cac cgg tcc cgggga ggc gga ggg gga tcc 259 Arg Gln Pro Pro Leu Pro His Arg Ser Arg GlyGly Gly Gly Gly Ser 40 45 50 cgc ggg ggc gcc cgg gcc tcg ccc gcc acg cagccg cca ccg ctg ctg 307 Arg Gly Gly Ala Arg Ala Ser Pro Ala Thr Gln ProPro Pro Leu Leu 55 60 65 ccg ccc tcg gcc acg ggt ccc gac gcg aca gtg ggcggg cca gcg ccg 355 Pro Pro Ser Ala Thr Gly Pro Asp Ala Thr Val Gly GlyPro Ala Pro 70 75 80 acc ccg ctg ctg ccc ccc tcg gcc aca gcc tcg gtc aagatg gag cca 403 Thr Pro Leu Leu Pro Pro Ser Ala Thr Ala Ser Val Lys MetGlu Pro 85 90 95 gag aac aag tac ctg ccc gaa ctc atg gcc gag aag gac tcgctc gac 451 Glu Asn Lys Tyr Leu Pro Glu Leu Met Ala Glu Lys Asp Ser LeuAsp 100 105 110 115 ccg tcc ttc act cac gcc atg cag ctg ctg acg gca gaaatt gag aag 499 Pro Ser Phe Thr His Ala Met Gln Leu Leu Thr Ala Glu IleGlu Lys 120 125 130 att cag aaa gga gac tca aaa aag gat gat gag gag aattac ttg gat 547 Ile Gln Lys Gly Asp Ser Lys Lys Asp Asp Glu Glu Asn TyrLeu Asp 135 140 145 tta ttt tct cat aag aac atg aaa ctg aaa gag cga gtgctg ata cct 595 Leu Phe Ser His Lys Asn Met Lys Leu Lys Glu Arg Val LeuIle Pro 150 155 160 gtc aag cag tat ccc aag ttc aat ttt gtg ggg aag attctt gga cca 643 Val Lys Gln Tyr Pro Lys Phe Asn Phe Val Gly Lys Ile LeuGly Pro 165 170 175 caa ggg aat aca atc aaa aga ctg cag gaa gag act ggtgca aag atc 691 Gln Gly Asn Thr Ile Lys Arg Leu Gln Glu Glu Thr Gly AlaLys Ile 180 185 190 195 tct gta ttg gga aag ggc tca atg aga gac aaa gccaag gag gaa gag 739 Ser Val Leu Gly Lys Gly Ser Met Arg Asp Lys Ala LysGlu Glu Glu 200 205 210 ctg cgc aaa ggt gga gac ccc aaa tat gcc cac ttgaat atg gat ctg 787 Leu Arg Lys Gly Gly Asp Pro Lys Tyr Ala His Leu AsnMet Asp Leu 215 220 225 cat gtc ttc att gaa gtc ttt gga ccc cca tgt gaggct tat gct ctt 835 His Val Phe Ile Glu Val Phe Gly Pro Pro Cys Glu AlaTyr Ala Leu 230 235 240 atg gcc cat gcc atg gag gaa gtc aag aaa ttt ctagta ccg gat atg 883 Met Ala His Ala Met Glu Glu Val Lys Lys Phe Leu ValPro Asp Met 245 250 255 atg gat gat atc tgt cag gag caa ttt cta gag ctgtcc tac ttg aat 931 Met Asp Asp Ile Cys Gln Glu Gln Phe Leu Glu Leu SerTyr Leu Asn 260 265 270 275 gga gta cct gaa ccc tct cgt gga cgt ggg gtgcca gtg aga ggc cgg 979 Gly Val Pro Glu Pro Ser Arg Gly Arg Gly Val ProVal Arg Gly Arg 280 285 290 gga gct gca cct cct cca cca cct gtt ccc aggggc cgt ggt gtt gga 1027 Gly Ala Ala Pro Pro Pro Pro Pro Val Pro Arg GlyArg Gly Val Gly 295 300 305 cca cct cgg ggg gct ttg gta cgt ggt aca ccagta agg gga gcc atc 1075 Pro Pro Arg Gly Ala Leu Val Arg Gly Thr Pro ValArg Gly Ala Ile 310 315 320 acc aga ggt gcc act gtg act cga ggc gtg ccaccc cca cct act gtg 1123 Thr Arg Gly Ala Thr Val Thr Arg Gly Val Pro ProPro Pro Thr Val 325 330 335 agg ggt gct cca gca cca aga gca cgg aca gcgggc atc cag agg ata 1171 Arg Gly Ala Pro Ala Pro Arg Ala Arg Thr Ala GlyIle Gln Arg Ile 340 345 350 355 cct ttg cct cca cct cct gca cca gaa acatat gaa gaa tat gga tat 1219 Pro Leu Pro Pro Pro Pro Ala Pro Glu Thr TyrGlu Glu Tyr Gly Tyr 360 365 370 gat gat aca tac gca gaa caa agt tac gaaggc tac gaa ggc tat tac 1267 Asp Asp Thr Tyr Ala Glu Gln Ser Tyr Glu GlyTyr Glu Gly Tyr Tyr 375 380 385 agc cag agt caa ggg gac tca gaa tat tatgac tat gga cat ggg gag 1315 Ser Gln Ser Gln Gly Asp Ser Glu Tyr Tyr AspTyr Gly His Gly Glu 390 395 400 gtt caa gat tct tat gaa gct tat ggc caggac gac tgg aat ggg acc 1363 Val Gln Asp Ser Tyr Glu Ala Tyr Gly Gln AspAsp Trp Asn Gly Thr 405 410 415 agg ccg tcg ctg aag gcc cct cct gct aggcca gtg aag gga gca tac 1411 Arg Pro Ser Leu Lys Ala Pro Pro Ala Arg ProVal Lys Gly Ala Tyr 420 425 430 435 aga gag cac cca tat gga cgt tattaaaaacaaa catgagggga aaatatcagt 1465 Arg Glu His Pro Tyr Gly Arg Tyr440 tatgagcaaa gttgttactg atttcttgta tctcccagga ttcctgttgc tttacccaca1525 acagacaagt aattgtctaa gtgtttttct tcgtggtccc cttcttctcc ccaccttatt1585 ccattcttaa ctctgcattc tggcttctgt atgtagtatt ttaaaatgag ttaaaataga1645 tttaggaata ttgaattaat tttttaagtg tgtagatgct tttttctttg ttgtttaaat1705 ataaacagaa gtgtaccttt tataataaaa aaaagaagtt gagtaaaaaa aaaaaacaca1765 caaacctgtt agtttcaaaa atgacattgc ttgcttaaag gttctgaagt aaaggcttgt1825 taagtttctc ttagttttga tttgaggcat cccgtaaagt tgtagttgca gaatcccaaa1885 ctaggctaca tttcaaaatt cagggctgtt taagatttaa aatcacaaac attaacggca1945 gtaggcacca ccatgtaaaa gtgagctcag acgtctctaa aaaatgtttc ctttataaaa2005 gcacatggcg gttgaatctt aaggttaaat tttaatatga aagatcctca tgaattaaat2065 agttgatgca atttttaacg ttaattgata taaaaaaaaa aacaacaaaa ttaggcttgt2125 aaaactgact ttttcattac gtgggttttg aaatctagcc ccagacatac tgtgttgaga2185 gatacttaga gggagggagt aggttttgaa gaggttgatg gtggtgggga gggaaggcct2245 cctgaattga gtttgatgca gagcttttta gccatgaaga atctttcagt catagtacta2305 ataattaaat tttcagtatt taaaaagaca aagtattttg tccatttgag attctgcact2365 ccatgaaaag ttcacttgga cgctggggcc aaaagctgtt gattttctta agttgacggt2425 tgtcaatata tcgaactgtt cccaagttag tcaagtatgt ctcaacacta gcatgatata2485 aaaagggaca ctgcagctga atgaaaaagg aatcaaaatc cactttgtac ataagttaaa2545 gtcctaattg gatttgtacc gtcctcccat tttgttctcg gaagattaaa tgctacatgt2605 gtaagtctgc ctaaataggt agcttaaact tatgtcaaaa tgtctgcagc agtttgtcaa2665 taaagtttag tcctttttta 2685 6 443 PRT Homo sapiens 6 Met Gln Arg ArgAsp Asp Pro Ala Ala Arg Met Ser Arg Ser Ser Gly 1 5 10 15 Arg Ser GlySer Met Asp Pro Ser Gly Ala His Pro Ser Val Arg Gln 20 25 30 Thr Pro SerArg Gln Pro Pro Leu Pro His Arg Ser Arg Gly Gly Gly 35 40 45 Gly Gly SerArg Gly Gly Ala Arg Ala Ser Pro Ala Thr Gln Pro Pro 50 55 60 Pro Leu LeuPro Pro Ser Ala Thr Gly Pro Asp Ala Thr Val Gly Gly 65 70 75 80 Pro AlaPro Thr Pro Leu Leu Pro Pro Ser Ala Thr Ala Ser Val Lys 85 90 95 Met GluPro Glu Asn Lys Tyr Leu Pro Glu Leu Met Ala Glu Lys Asp 100 105 110 SerLeu Asp Pro Ser Phe Thr His Ala Met Gln Leu Leu Thr Ala Glu 115 120 125Ile Glu Lys Ile Gln Lys Gly Asp Ser Lys Lys Asp Asp Glu Glu Asn 130 135140 Tyr Leu Asp Leu Phe Ser His Lys Asn Met Lys Leu Lys Glu Arg Val 145150 155 160 Leu Ile Pro Val Lys Gln Tyr Pro Lys Phe Asn Phe Val Gly LysIle 165 170 175 Leu Gly Pro Gln Gly Asn Thr Ile Lys Arg Leu Gln Glu GluThr Gly 180 185 190 Ala Lys Ile Ser Val Leu Gly Lys Gly Ser Met Arg AspLys Ala Lys 195 200 205 Glu Glu Glu Leu Arg Lys Gly Gly Asp Pro Lys TyrAla His Leu Asn 210 215 220 Met Asp Leu His Val Phe Ile Glu Val Phe GlyPro Pro Cys Glu Ala 225 230 235 240 Tyr Ala Leu Met Ala His Ala Met GluGlu Val Lys Lys Phe Leu Val 245 250 255 Pro Asp Met Met Asp Asp Ile CysGln Glu Gln Phe Leu Glu Leu Ser 260 265 270 Tyr Leu Asn Gly Val Pro GluPro Ser Arg Gly Arg Gly Val Pro Val 275 280 285 Arg Gly Arg Gly Ala AlaPro Pro Pro Pro Pro Val Pro Arg Gly Arg 290 295 300 Gly Val Gly Pro ProArg Gly Ala Leu Val Arg Gly Thr Pro Val Arg 305 310 315 320 Gly Ala IleThr Arg Gly Ala Thr Val Thr Arg Gly Val Pro Pro Pro 325 330 335 Pro ThrVal Arg Gly Ala Pro Ala Pro Arg Ala Arg Thr Ala Gly Ile 340 345 350 GlnArg Ile Pro Leu Pro Pro Pro Pro Ala Pro Glu Thr Tyr Glu Glu 355 360 365Tyr Gly Tyr Asp Asp Thr Tyr Ala Glu Gln Ser Tyr Glu Gly Tyr Glu 370 375380 Gly Tyr Tyr Ser Gln Ser Gln Gly Asp Ser Glu Tyr Tyr Asp Tyr Gly 385390 395 400 His Gly Glu Val Gln Asp Ser Tyr Glu Ala Tyr Gly Gln Asp AspTrp 405 410 415 Asn Gly Thr Arg Pro Ser Leu Lys Ala Pro Pro Ala Arg ProVal Lys 420 425 430 Gly Ala Tyr Arg Glu His Pro Tyr Gly Arg Tyr 435 4407 1894 DNA Homo sapiens CDS (12)..(1322) 7 cggaaaaaaa a atg gtt gaa gcagat cgc cca gga aag ctc ttc att ggt 50 Met Val Glu Ala Asp Arg Pro GlyLys Leu Phe Ile Gly 1 5 10 ggg ctt aat acg gaa aca aat gag aaa gct cttgaa gca gta ttt ggc 98 Gly Leu Asn Thr Glu Thr Asn Glu Lys Ala Leu GluAla Val Phe Gly 15 20 25 aaa tat gga cga ata gtg gaa gta ctc ttg atg aaagac cgt gaa acc 146 Lys Tyr Gly Arg Ile Val Glu Val Leu Leu Met Lys AspArg Glu Thr 30 35 40 45 aac aaa tca aga gga ttt gct ttt gtc acc ttt gaaagc cca gca gac 194 Asn Lys Ser Arg Gly Phe Ala Phe Val Thr Phe Glu SerPro Ala Asp 50 55 60 gct aag gat gca gcc aga gac atg aat gga aag tca ttagat gga aaa 242 Ala Lys Asp Ala Ala Arg Asp Met Asn Gly Lys Ser Leu AspGly Lys 65 70 75 gcc atc aag gtg gaa caa gcc acc aaa cca tca ttt gaa agtggt aga 290 Ala Ile Lys Val Glu Gln Ala Thr Lys Pro Ser Phe Glu Ser GlyArg 80 85 90 cgt gga ccg cct cca cct cca aga agt aga ggc cct cca aga ggtctt 338 Arg Gly Pro Pro Pro Pro Pro Arg Ser Arg Gly Pro Pro Arg Gly Leu95 100 105 aga ggt gga aga gga gga agt gga gga acc agg gga cct ccc tcacgg 386 Arg Gly Gly Arg Gly Gly Ser Gly Gly Thr Arg Gly Pro Pro Ser Arg110 115 120 125 gga gga cac atg gat gac ggt gga tat tcc atg aat ttt aacatg agt 434 Gly Gly His Met Asp Asp Gly Gly Tyr Ser Met Asn Phe Asn MetSer 130 135 140 tct tcc agg gga cca ctc cca gta aaa aga gga cca cca ccaaga agt 482 Ser Ser Arg Gly Pro Leu Pro Val Lys Arg Gly Pro Pro Pro ArgSer 145 150 155 ggg ggt cct cct cct aag aga tct gca cct tca gga cca gttcgc agt 530 Gly Gly Pro Pro Pro Lys Arg Ser Ala Pro Ser Gly Pro Val ArgSer 160 165 170 agc agt gga atg gga gga aga gct cct gta tca cgt gga agagat agt 578 Ser Ser Gly Met Gly Gly Arg Ala Pro Val Ser Arg Gly Arg AspSer 175 180 185 tat gga ggt cca cct cga agg gaa ccg ctg ccc tct cgt agagat gtt 626 Tyr Gly Gly Pro Pro Arg Arg Glu Pro Leu Pro Ser Arg Arg AspVal 190 195 200 205 tat ttg tct cca aga gat gat ggg tat tct act aaa gacagc tat tca 674 Tyr Leu Ser Pro Arg Asp Asp Gly Tyr Ser Thr Lys Asp SerTyr Ser 210 215 220 agc aga gat tac cca agt tct cgt gat act aga gat tatgca cca cca 722 Ser Arg Asp Tyr Pro Ser Ser Arg Asp Thr Arg Asp Tyr AlaPro Pro 225 230 235 cca cga gat tat act tac cgt gat tat ggt cat tcc agttca cgt gat 770 Pro Arg Asp Tyr Thr Tyr Arg Asp Tyr Gly His Ser Ser SerArg Asp 240 245 250 gac tat cca tca aga gaa tat agc gat aga gat gga tatggt cgt gat 818 Asp Tyr Pro Ser Arg Glu Tyr Ser Asp Arg Asp Gly Tyr GlyArg Asp 255 260 265 cgt gac tat tca gat cat cca agt gga ggt tcc tac agagat tca tat 866 Arg Asp Tyr Ser Asp His Pro Ser Gly Gly Ser Tyr Arg AspSer Tyr 270 275 280 285 gag agt tat ggt aac tca cgt agt gct cca cct acacga ggg ccc ccg 914 Glu Ser Tyr Gly Asn Ser Arg Ser Ala Pro Pro Thr ArgGly Pro Pro 290 295 300 cca tct tat ggt gga agc agt cgc tat gat gat tacagc agc tca cgt 962 Pro Ser Tyr Gly Gly Ser Ser Arg Tyr Asp Asp Tyr SerSer Ser Arg 305 310 315 gac gga tat ggt gga agt cga gac agt tac tca agcagc cga agt gat 1010 Asp Gly Tyr Gly Gly Ser Arg Asp Ser Tyr Ser Ser SerArg Ser Asp 320 325 330 ctc tac tca agt ggt cgt gat cgg gtt ggc aga caagaa aga ggg ctt 1058 Leu Tyr Ser Ser Gly Arg Asp Arg Val Gly Arg Gln GluArg Gly Leu 335 340 345 ccc cct tct atg gaa agg ggg tac ctc ctc cac gtgatt cct aca gca 1106 Pro Pro Ser Met Glu Arg Gly Tyr Leu Leu His Val IlePro Thr Ala 350 355 360 365 gtt caa gcc gcg gac gac caa gag gtg gtg gccgtg gag gaa gcc gat 1154 Val Gln Ala Ala Asp Asp Gln Glu Val Val Ala ValGlu Glu Ala Asp 370 375 380 ctg ata gag ggg gag gca gaa gca gat act agaaac aaa caa aac ttt 1202 Leu Ile Glu Gly Glu Ala Glu Ala Asp Thr Arg AsnLys Gln Asn Phe 385 390 395 gga cca aaa tcc cag ttc aaa gaa aca aaa agtgga aac tat tct atc 1250 Gly Pro Lys Ser Gln Phe Lys Glu Thr Lys Ser GlyAsn Tyr Ser Ile 400 405 410 ata act acc caa gga cta cta aaa gga aaa attgtg tta ctt ttt tta 1298 Ile Thr Thr Gln Gly Leu Leu Lys Gly Lys Ile ValLeu Leu Phe Leu 415 420 425 aat tcc ctg tta agt tcc cct cca taatttttatgttcttgtga ggaaaaaagt 1352 Asn Ser Leu Leu Ser Ser Pro Pro 430 435aaaacatgtt taattttatt tgacttctgc attgcttttc aacaagcaaa tgttaaatgt 1412gttaagactt gtactagtgt tgtaactttc caagtaaaag tatcccctaa aggccacttc 1472ctatctgatt tttcccagca aatgaggcag gcaattctag tcttccacaa aacatctagc 1532catctaaaat ggagagatga atcattctac ctatacaaac aagctagcta ttagagggtg 1592gttggggtat gctactcata agatttcagg gtgtcttcca actgaaatct caatgttctc 1652agtacgaaaa acctgaaatc acatgcctat gtaaggaaag tgctattcac ccagtaaacc 1712caaaaaagca aatggataat gctggccatt ttgcctttct gacatttcct tgggaatctg 1772caagaacctc ccctttccct tcccccaata agaccattta agtgtgtgtt aaacaactac 1832agaatactaa gtaaaaagtt tggccaaaac caaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1892aa 1894 8 437 PRT Homo sapiens 8 Met Val Glu Ala Asp Arg Pro Gly Lys LeuPhe Ile Gly Gly Leu Asn 1 5 10 15 Thr Glu Thr Asn Glu Lys Ala Leu GluAla Val Phe Gly Lys Tyr Gly 20 25 30 Arg Ile Val Glu Val Leu Leu Met LysAsp Arg Glu Thr Asn Lys Ser 35 40 45 Arg Gly Phe Ala Phe Val Thr Phe GluSer Pro Ala Asp Ala Lys Asp 50 55 60 Ala Ala Arg Asp Met Asn Gly Lys SerLeu Asp Gly Lys Ala Ile Lys 65 70 75 80 Val Glu Gln Ala Thr Lys Pro SerPhe Glu Ser Gly Arg Arg Gly Pro 85 90 95 Pro Pro Pro Pro Arg Ser Arg GlyPro Pro Arg Gly Leu Arg Gly Gly 100 105 110 Arg Gly Gly Ser Gly Gly ThrArg Gly Pro Pro Ser Arg Gly Gly His 115 120 125 Met Asp Asp Gly Gly TyrSer Met Asn Phe Asn Met Ser Ser Ser Arg 130 135 140 Gly Pro Leu Pro ValLys Arg Gly Pro Pro Pro Arg Ser Gly Gly Pro 145 150 155 160 Pro Pro LysArg Ser Ala Pro Ser Gly Pro Val Arg Ser Ser Ser Gly 165 170 175 Met GlyGly Arg Ala Pro Val Ser Arg Gly Arg Asp Ser Tyr Gly Gly 180 185 190 ProPro Arg Arg Glu Pro Leu Pro Ser Arg Arg Asp Val Tyr Leu Ser 195 200 205Pro Arg Asp Asp Gly Tyr Ser Thr Lys Asp Ser Tyr Ser Ser Arg Asp 210 215220 Tyr Pro Ser Ser Arg Asp Thr Arg Asp Tyr Ala Pro Pro Pro Arg Asp 225230 235 240 Tyr Thr Tyr Arg Asp Tyr Gly His Ser Ser Ser Arg Asp Asp TyrPro 245 250 255 Ser Arg Glu Tyr Ser Asp Arg Asp Gly Tyr Gly Arg Asp ArgAsp Tyr 260 265 270 Ser Asp His Pro Ser Gly Gly Ser Tyr Arg Asp Ser TyrGlu Ser Tyr 275 280 285 Gly Asn Ser Arg Ser Ala Pro Pro Thr Arg Gly ProPro Pro Ser Tyr 290 295 300 Gly Gly Ser Ser Arg Tyr Asp Asp Tyr Ser SerSer Arg Asp Gly Tyr 305 310 315 320 Gly Gly Ser Arg Asp Ser Tyr Ser SerSer Arg Ser Asp Leu Tyr Ser 325 330 335 Ser Gly Arg Asp Arg Val Gly ArgGln Glu Arg Gly Leu Pro Pro Ser 340 345 350 Met Glu Arg Gly Tyr Leu LeuHis Val Ile Pro Thr Ala Val Gln Ala 355 360 365 Ala Asp Asp Gln Glu ValVal Ala Val Glu Glu Ala Asp Leu Ile Glu 370 375 380 Gly Glu Ala Glu AlaAsp Thr Arg Asn Lys Gln Asn Phe Gly Pro Lys 385 390 395 400 Ser Gln PheLys Glu Thr Lys Ser Gly Asn Tyr Ser Ile Ile Thr Thr 405 410 415 Gln GlyLeu Leu Lys Gly Lys Ile Val Leu Leu Phe Leu Asn Ser Leu 420 425 430 LeuSer Ser Pro Pro 435 9 323 DNA Homo sapiens 9 gctatagcag aaccgctggggtaacaacaa ccgggataac aacaactcca acaacagagg 60 cagctacaac cgggctccccagcaacagcc gccaccacag cagcctccgc caccacagcc 120 accaccccag cagccaccgccaccacccag ctacagccct gctcggaacc ccccaggggc 180 cagcacctac aataagaacagcaacatccc tggctcaagc gccaatacca gcacccccac 240 cgtcagcagc tacagcccttccacagccga gttacagcca gccaccctac anccagggga 300 ggttacagcc agggttacacagg 323 10 914 DNA Homo sapiens 10 ggcggcttcc agaaaaaagg ggaggcagcggtggaggagg caactaccga ggaggtttca 60 accgcagcgg aggtggtggc tatagcagaaccgctggggt aacaacaacc gggataacaa 120 caactccaac aacagaggca gctacaaccgggctccccag caacagccgc caccacagca 180 gcctccgcca ccacagccac caccccagcagccaccgcca ccacccagct acagccctgc 240 tcggaacccc ccaggggcca gcacctacaataagaacagc aacatccctg gctcaagcgc 300 caataccagc acccccaccg tcagcagctacagcccttcc acagccgagt tacagccagc 360 caccctacaa ccaggggagg ttacagccagggttacacag gcccaccgcc tccacctcca 420 ccaccacctg cctacaacta tgggagctacggcggttaca acccggcccc ctatacccca 480 ccgccacccc ccaccgcaca gacctaccctcagcccaact ataaccagta tcagcagtat 540 gccagcagtg gaaccagtac tatcagaaccagggccagtg gcgccatact acgggaacta 600 cgactacggg agctactccg ggaacacacagggtggcaca agtacacagt agccagtgtg 660 acccagaggc tcccggaggc ccctgccggcttcctccacc agcgcctgcc tcggcccctc 720 ctctgccccc gccagatccc gtggtgctggggatggggtc atcccagggc tgcctccctc 780 cagcccactg cctcccctct gaggggcttccttcccctcc atagggccag gcattttttt 840 ctggattcaa acaggcaaca atgaccttttattttctgtt tgtccccacc tccccagcct 900 tccacctcct gttc 914 11 45 PRT Homosapiens 11 Arg Leu Pro Glu Lys Arg Gly Gly Ser Gly Gly Gly Gly Asn TyrArg 1 5 10 15 Gly Gly Phe Asn Arg Ser Gly Gly Gly Gly Tyr Ser Arg ThrAla Gly 20 25 30 Val Thr Thr Thr Gly Ile Thr Thr Thr Pro Thr Thr Glu 3540 45 12 70 PRT Homo sapiens 12 Met Gly Ala Thr Ala Val Thr Thr Arg ProPro Ile Pro His Arg His 1 5 10 15 Pro Pro Pro His Arg Pro Thr Leu SerPro Thr Ile Thr Ser Ile Ser 20 25 30 Ser Met Pro Ala Val Glu Pro Val LeuSer Glu Pro Gly Pro Val Ala 35 40 45 Pro Tyr Tyr Gly Asn Tyr Asp Tyr GlySer Tyr Ser Gly Asn Thr Gln 50 55 60 Gly Gly Thr Ser Thr Gln 65 70 13 33PRT Homo sapiens 13 Gly Gly Phe Gln Lys Lys Gly Glu Ala Ala Val Glu GluAla Thr Thr 1 5 10 15 Glu Glu Val Ser Thr Ala Ala Glu Val Val Ala IleAla Glu Pro Leu 20 25 30 Gly 14 44 DNA Artificial Sequence Descriptionof Artificial Sequenceforward primer 14 ggactaggcc gaggtggcct ctccaggccttgattatgag cctg 44 15 46 DNA Artificial Sequence Description ofArtificial Sequencereverse primer 15 ggactaggcc tcctcggccc tacctctgcactatgtcact gatttc 46

What is claimed is:
 1. A method for identifying a molecule thatmodulates the formation of a complex comprising 53BP2 and a proteinselected from the group consisting of β-tubulin, p62, hnRNP G, the53BP2-IP1 polypeptide comprising SEQ ID NO: 11, the 53BP2-IP2polypeptide comprising SEQ ID NO:12, and the 53BP2-IP3 polypeptidecomprising SEQ ID NO:13; said method comprising contacting one or morecandidate molecules with 53BP2 in the presence of said selected protein;and measuring the amount of complex that forms between 53BP2 and saidselected protein; wherein an increase or decrease in the amount ofcomplex that forms relative to the amount that forms in the absence ofthe candidate molecules indicates that the candidate molecules modulatethe formation of said complex comprising 53BP2 and said selectedprotein.
 2. The method of claim 1, wherein said contacting is carriedout by contacting the candidate molecule with an isolated recombinantcell comprising a 53BP2 gene and a 53BP2-IP gene selected from the groupconsisting of β-tubulin, p62, hnRNP G, 53BP2-IP1, 53BP2-IP2, and53BP2-IP3, wherein the 53BP2 gene is under the control of a promoterthat is not the native 53BP2 gene promoter and the 53BP2-IP gene isunder the control of a promoter that is not the native 53BP2-IP genepromoter.
 3. The method of claim 2, wherein said isolated recombinantcell is an animal cell.
 4. The method of claim 2, wherein said isolatedrecombinant cell is a mammalian cell.
 5. The method of claim 1 whereinthe candidate molecule is recombinantly expressed in an isolatedrecombinant cell comprising a 53BP2 gene and a 53BP2-IP gene selectedfrom the group consisting of β-tubulin, p62, hnRNP G, 53BP2-IP1,53BP2-IP2, and 53BP2-IP3, wherein the 53BP2 gene is under the control ofa promoter that is not the native 53BP2 gene promoter and the 53BP2-IPgene is under the control of a promoter that is not the native 53BP2-IPgene promoter.
 6. The method of claim 5, wherein said isolatedrecombinant cell is an animal cell.
 7. The method of claim 5, whereinsaid isolated recombinant cell is a mammalian cell.
 8. The method ofclaim 1 wherein said contacting is carried out in vitro.
 9. A method foridentifying a molecule that modulates the transcriptional regulatoryactivity of a complex comprising 53BP2 and a protein selected from thegroup consisting of β-tubulin, p62, hnRNP G, the 53BP2-IP1 polypeptidecomprising SEQ ID NO: 11, the 53BP2-IP2 polypeptide comprising SEQ IDNO:12, and the 53BP2-IP3 polypeptide comprising SEQ ID NO:13; saidmethod comprising contacting one or more candidate molecules with saidcomplex; and measuring the transcriptional regulatory activity of saidcomplex; wherein an increase or decrease in the transcriptionalregulatory activity of said complex compared to the transcriptionalregulatory activity of said complex in the absence of the candidatemolecule indicates that the candidate molecule modulates thetranscriptional regulatory activity of said complex.
 10. The method ofclaim 9, wherein said contacting is carried out by contacting thecandidate molecule with an isolated recombinant cell comprising a53BP2-gene and a 53BP2-IP gene selected from the group consisting ofβ-tubulin, p62, hnRNP G, 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3, in whichthe 53BP2 gene is under the control of a promoter is not the native53BP2 gene promoter and the 53BP2-IP gene is under the control of apromoter that is not the native 53BP2-IP gene promoter.
 11. The methodof claim 10, wherein said isolated recombinant cell is an animal cell.12. The method of claim 10, wherein said isolated recombinant cell is amammalian cell.
 13. The method of claim 9 wherein the candidate moleculeis recombinantly expressed in an isolated recombinant cell comprising a53BP2 gene and a 53BP2-IP gene selected from the group consisting ofβ-tubulin, p62, hnRNP G, 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3, in whichthe 53BP2 gene is under the control of a promoter that is not the native53BP2 gene promoter and the 53BP2-IP gene is under the control of apromoter that is not the native 53BP2-IP gene promoter.
 14. The methodof claim 13, wherein said isolated recombinant cell is an animal cell.15. The method of claim 13, wherein said isolated recombinant cell is amammalian cell.
 16. The method of claim 9 wherein said contact iscarried out in vitro.